Substituted pyridine compounds for inflammation and immune-related uses

ABSTRACT

The invention relates to certain compounds of Formula (I): 
     
       
         
         
             
             
         
       
         
         
           
             or pharmaceutically acceptable salts, solvates, clathrates, or prodrugs thereof, that are useful as immunosuppressive agents and for treating and preventing inflammatory conditions, allergic disorders, and immune disorders.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/194,831, filed Oct. 1, 2008, the entire disclosure of which isincorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to biologically active chemical compounds thatmay be used for immunosuppression or to treat or prevent inflammatoryconditions and immune disorders.

BACKGROUND OF THE INVENTION

Inflammation is a mechanism that protects mammals from invadingpathogens. However, while transient inflammation is necessary to protecta mammal from infection, uncontrolled inflammation causes tissue damageand is the underlying cause of many illnesses. Inflammation is typicallyinitiated by binding of an antigen to T-cell antigen receptor. Antigenbinding by a T-cell initiates calcium influx into the cell via calciumion channels, such as Ca²⁺-release-activated Ca²⁺ channels (CRAC).Calcium ion influx in turn initiates a signaling cascade that leads toactivation of these cells and an inflammatory response characterized bycytokine production.

Interleukin 2 (IL-2) is a cytokine that is secreted by T cells inresponse to calcium ion influx into the cell. IL-2 modulatesimmunological effects on many cells of the immune system. For example,it is a potent T cell mitogen that is required for the T cellproliferation, promoting their progression from G1 to S phase of thecell cycle; it stimulates the growth of NK cells; and it acts as agrowth factor to B cells and stimulates antibody synthesis.

IL-2, although useful in the immune response, can cause a variety ofproblems. IL-2 damages the blood-brain barrier and the endothelium ofbrain vessels. These effects may be the underlying causes ofneuropsychiatric side effects observed under IL-2 therapy, e.g.,fatigue, disorientation and depression. It also alters theelectrophysiological behavior of neurons.

Due to its effects on both T and B cells, IL-2 is a major centralregulator of immune responses. It plays a role in inflammatoryreactions, tumor surveillance, and hematopoiesis. It also affects theproduction of other cytokines, inducing IL-1, TNFα and TNF-β secretion,as well as stimulating the synthesis of IFN-γ in peripheral leukocytes.

T cells that are unable to produce IL-2 become inactive (anergic). Thisrenders them potentially inert to any antigenic stimulation they mightreceive in the future. As a result, agents which inhibit IL-2 productioncan be used for immunosuppression or to treat or prevent inflammationand immune disorders. This approach has been clinically validated withimmunosuppressive drugs such as cyclosporin, FK506, and RS61443. Despitethis proof of concept, agents that inhibit IL-2 production remain farfrom ideal. Among other problems, efficacy limitations and unwanted sideeffects (including dose-dependant nephrotoxicity and hypertension)hinder their use.

Over-production of proinflammatory cytokines other than IL-2 has alsobeen implicated in many autoimmune diseases. For example, Interleukin 5(IL-5), a cytokine that increases the production of eosinophils, isincreased in asthma. Overproduction of IL-5 is associated withaccumulation of eosinophils in the asthmatic bronchial mucosa, ahallmark of allergic inflammation. Thus, patients with asthma and otherinflammatory disorders involving the accumulation of eosinophils wouldbenefit from the development of new drugs that inhibit the production ofIL-5.

Interleukin 4 (IL-4) and interleukin 13 (IL-13) have been identified asmediators of the hypercontractility of smooth muscle found ininflammatory bowel disease and asthma. Thus, patients with asthma andinflammatory bowel disease would benefit from the development of newdrugs that inhibit IL-4 and IL-13 production.

Granulocyte macrophage-colony stimulating factor (GM-CSF) is a regulatorof maturation of granulocyte and macrophage lineage population and hasbeen implicated as a key factor in inflammatory and autoimmune diseases.Anti-GM-CSF antibody blockade has been shown to ameliorate autoimmunedisease. Thus, development of new drugs that inhibit the production ofGM-CSF would be beneficial to patients with an inflammatory orautoimmune disease.

SUMMARY OF THE INVENTION

The present disclosure, in an aspect, addresses the continuing need fornew drugs which overcome one or more of the shortcomings of drugscurrently used for immunosuppression or in the treatment or preventionof inflammatory disorders, allergic disorders and autoimmune disorders.Desirable properties of such drugs include efficacy against diseases ordisorders that are currently untreatable or poorly treatable, newmechanism of action, oral bioavailability and/or reduced side effects.Accordingly, compounds that inhibit the activity of CRAC ion channelsand inhibit the production of IL-2, IL-4, IL-5, IL-13, GM-CSF, TNFα, andIFN-γ are disclosed herein. These compounds are particularly useful forimmunosuppression and/or to treat or prevent inflammatory conditions andimmune disorders. The particular genus of compounds described herein areparticularly advantageous in that they are believed to combineinhibition of CRAC ion channels (e.g., as measured by modulated I_(CRAC)current) and cytokines including IL-2, low incidence of off-targeteffects, and a favorable toxicity profile.

The invention features compounds of formula (I):

or pharmaceutically acceptable salt thereof; wherein:

each of X₁ and X₂ is independently N, C or N⁺O⁻;

Y₁ is S or CH;

Y₂ is S, N, or CH;

Z is a bond or a linker having 1-6 atoms;

Y₃ is CH₂ or C═O;

R¹ is heteroaryl, aryl, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,wherein each substituent represented by R¹ is independently andoptionally substituted with one to three halo, (C₁-C₄)alkyl,(C₂-C₄)alkenyl, (C₂-C₄)alkynyl, CORE, COOR⁶, CON(R⁶)₂, N(R⁶)₂,NR⁶CON(R⁶)₂, NR⁶CSN(R⁶)₂, OR⁶, S(O)_(p)R⁶, S(O)_(p)N(R⁶)₂, CN, NO₂, orN₃;

R² is (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, heteroaryl,heteroaryl(C₁-C₂)alkyl, heteroaryl(C₂)alkenyl, heteroaryl(C₂)alkynyl,aryl(C₁-C₂)alkyl, aryl(C₂)alkenyl, aryl(C₂)alkynyl, CN, COR⁶, COOR⁶,CON(R⁶)₂, CSR⁶, CSOR⁶, or CSN(R⁶)₂, wherein each substituent representedby R² is independently and optionally substituted with one to threehalo, (C₁-C₄)alkyl, (C₂-C₄)alkenyl, (C₂-C₄)alkynyl, COR⁶, COOR⁶,CON(R⁶)₂, N(R⁶)₂, NR⁶CON(R⁶)₂, NR⁶CSN(R⁶)₂, OR⁶, SR⁶, CN, NO₂, or N₃;

R³ is H, halo, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, aryl,heteroaryl, (C₃-C₇)cycloalkyl, heterocycloalkyl, COR⁶, COOR⁶, CON(R⁶)₂,N(R⁶)₂, NR⁶CON(R⁶)₂, NR⁶CSN(R⁶)₂, OR⁶, S(O)_(p)R⁶, CN, NO₂,S(O)_(p)N(R⁶)₂ or N₃, wherein each substituent represented by R³ havinga hydrogen atom is optionally and independently substituted with halo,(C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₁-C₆)alkoxy, (C₁-C₆)haloalkoxy,(C₁-C₆)hydroxyalkyl, —OH, —NH₂, NH(C₁-C₃)alkyl, N((C₁-C₃)alkyl)₂, or CN;

R⁴ is H, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, heteroaryl,heteroaryl(C₁-C₂)alkyl, heteroaryl(C₁-C₂)alkenyl, heteroaryl(C₂)alkynyl,aryl, aryl(C₁-C₂)alkyl, aryl(C₂)alkenyl, aryl(C₂)alkynyl,(C₃-C₇)cycloalkyl, heterocycloalkyl, OR⁶, or CON(R⁶)₂;

each R⁵ is independently halo, (C₁-C₆)alkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, heteroaryl, heteroaryl(C₁-C₂)alkyl,heteroaryl(C₂)alkenyl, heteroaryl(C₂)alkynyl, (C₃-C₇)cycloalkyl,heterocycloalkyl, aryl, aryl(C₁-C₂)alkyl, aryl(C₂)alkenyl,aryl(C₂)alkynyl, (C₁-C₆)haloalkyl, COR⁶, COOR⁶, CON(R⁶)₂, N(R⁶)₂,NR⁶CON(R⁶)₂, NR⁶CSN(R⁶)₂, OR⁶, S(O)_(p)R⁶, CN, NO₂, or N₃, wherein eachsubstituent represented by R⁵ having a hydrogen atom is optionally anindependently substituted with halo, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl,(C₁-C₆)alkoxy, (C₁-C₆)haloalkoxy, (C₁-C₆)hydroxyalkyl, aryl, heteroaryl,(C₃-C₇)cycloalkyl, heterocycloalkyl, —OH, —NH₂, NH(C₁-C₃)alkyl,N((C₁-C₃)alkyl)₂, or CN;

each R⁶ is independently H, (C₁-C₆)alkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, (C₃-C₇)cycloalkyl, heterocycloalkyl, heteroaryl,heteroaryl(C₁-C₂)alkyl, heteroaryl(C₂)alkenyl, heteroaryl(C₂)alkynyl,aryl, aryl(C₁-C₂)alkyl, aryl(C₂)alkenyl, or aryl(C₂)alkynyl or two R⁶substituents attached to the same or adjacent atoms are taken togetherto form a heterocycloalkyl or heteroaryl;

n is 0-5; and

p is 0-2;

wherein one but not both of Y₁ and Y₂ is S; and one of the dashed bondsis a double bond, and one is a single bond.

In certain embodiments, Z is (C₁-C₆)alkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, heteroaryl, CO, COO, CON(R⁶), CS, CSO, or CSN(R⁶), e.g.,CH₂, CH₂CH₂, CO, thiazolyl, or In alternative embodiments, Z is a bond.In another embodiment, Z may be a bond, Y₁ may be S, and Y₂ may be N,e.g., when R¹ is heteroaryl or aryl, e.g., pyridinyl, and Y³ is C═O.

In certain embodiments, R¹ is heteroaryl, e.g., pyridinyl, pyrazolyl,oxazolyl, imidazolyl, or tetrazolyl, each of which may be N-substitutedwith (C₁-C₆)alkyl, which may in turn be substituted with N(R⁶)₂; R² is(C₁-C₆)alkyl, (C₁-C₆)alkenyl, or heteroaryl, each of which is optionallysubstituted with one to three halo, (C₁-C₄)alkyl, (C₁-C₄)alkenyl,(C₁-C₄)alkynyl, CORE, COOR⁶, CON(R⁶)₂, N(R⁶)₂, NR⁶CON(R⁶)₂, NR⁶CSN(R⁶)₂,OR⁶, SR⁶, CN, NO₂, or N₃; R³ and R⁴ are H, and/or n is 2, and R⁵ ishalo.

X₁ and X₂ are C; X₁ is N, and X₂ is N; X₁ is C, and X₂ is N; or X₁ is N,and X₂ is C.

Y₁ is S, and Y₂ is CH; Y₁ is S, and Y₂ is N; or Y₁ is CH, and Y₂ is S.

In another embodiment compounds of formula (II):

or pharmaceutically acceptable salts thereof are described, wherein:

one of X₁ and X₂ is N provided that the other is C;

Y₁ is S, N or CH;

Z is a bond; a —CH₂— group; a C₂ alkylene, alkenylene, or alkynylenegroup; or a carbonyl group;

R¹ is heteroaryl, aryl, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, or (C₂-C₆)alkynyl,and is optionally substituted with one to three halo, (C₁-C₄)alkyl,(C₂-C₄)alkenyl, (C₂-C₄)alkynyl, COR⁶, COOR⁶, CON(R⁶)₂, N(R⁶)₂,NR⁶CON(R⁶)₂, NR⁶CSN(R⁶)₂, OR⁶, S(O)_(p)R⁶, S(O)_(p)N(R⁶)₂, CN, NO₂, orN₃;

R² is (C₁-C₆)alkyl, (C₂-C₆)alkenyl, or (C₂-C₆)alkynyl, and is optionallysubstituted with one to three halo, (C₁-C₄)alkyl, (C₂-C₄)alkenyl,(C₂-C₄)alkynyl, COR⁶, COOR⁶, CON(R⁶)₂, N(R⁶)₂, NR⁶CON(R⁶)₂, NR⁶CSN(R⁶)₂,OR⁶, SR⁶, CN, NO₂, or N₃;

R³ is H, halo, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, COR⁶,COOR⁶, CON(R⁶)₂, N(R⁶)₂, NR⁶CON(R⁶)₂, NR⁶CSN(R⁶)₂, OR⁶, S(O)_(p)R⁶, CN,NO₂, S(O)_(p)N(R⁶)₂ or N₃, and is optionally and independentlysubstituted with halo, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₁-C₆)alkoxy,(C₁-C₆)haloalkoxy, (C₁-C₆)hydroxyalkyl, —OH, —NH₂, NH(C₁-C₃)alkyl,N((C₁-C₃)alkyl)₂, or CN;

R⁴ is H, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, OR⁶, or CON(R⁶)₂;

each R⁵ is independently halo, (C₁-C₆)alkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, COR⁶, COOR⁶, CON(R⁶)₂, N(R⁶)₂, NR⁶CON(R⁶)₂, NR⁶CSN(R⁶)₂,OR⁶, S(O)_(p)R⁶, CN, NO₂, or N₃, and is optionally substituted withhalo, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₁-C₆)alkoxy, (C₁-C₆)haloalkoxy,(C₁-C₆)hydroxyalkyl, —OH, —NH₂, NH(C₁-C₃)alkyl, N((C₁-C₃)alkyl)₂, or CN;

each R⁶ is independently H, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, or(C₂-C₆)alkynyl;

n is 0, 1, 2, 3, 4 or 5; and

p is 0, 1 or 2.

In embodiments of formula (II), when Z is a bond, Y₂ is CH, one of X₁and X₂ is N, and the other of X₁ and X₂ is C, and R² is methyl, then R¹is other than 1,3-oxazol-5-yl; and when Z is a bond, Y₂ is CH, one of X₁and X₂ is N, and the other of X₁ and X₂ is C, and R² is methyl, then R¹is other than 1-methyl-1H-imidazol-5-yl, 1,3-oxazol-5-yl, isoxazol-5-yl,1,3-thiazol-2-yl and 1H-imidazol-5-yl. In other embodiments, R¹ has onesubstituent; Z is a carbonyl group or a bond; Z is a bond or a C₂alkylene, alkenylene, or alkynylene group; R¹ is pyridyl or pyrazolyl;X₁ is C, X₂ is N; Y₂ is N; R² is C₁-C₃ alkyl; R³ is H; R⁴ is H; n is 2;and R⁵ is halo; R¹ is pyridyl, pyrazolyl or tetrazolyl; X₁ and X₂ are C;Y₂ is C; R³ is H; R⁴ is H; n is 2; and R⁵ is halo; R¹ is heteroaryl; R¹is pyridinyl, pyrazolyl, oxazolyl, imidazolyl, or tetrazolyl, each ofwhich may be N-substituted with (C₁-C₆)alkyl, which may in turn besubstituted with N(R⁶)₂. R³ and R⁴ are H; n is 2, and R⁵ is halo; and X₁is C, and X₂ is N; X₁ is N, and X₂ is C.

In another embodiment, compounds of formula (III):

or pharmaceutically acceptable salts thereof are described, wherein:

Y₁ is S, N or CH;

Z is a bond; a —CH₂— group; a C₂ alkylene, alkenylene, or alkynylenegroup; or a carbonyl group;

R¹— is heteroaryl, aryl, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, or(C₂-C₆)alkynyl, and is optionally substituted with one to three halo,(C₁-C₄)alkyl, (C₂-C₄)alkenyl, (C₂-C₄)alkynyl, COR⁶, COOR⁶, CON(R⁶)₂,N(R⁶)₂, NR⁶CON(R⁶)₂, NR⁶CSN(R⁶)₂, OR⁶, S(O)_(p)R⁶, S(O)_(p)N(R⁶)₂, CN,NO₂, or N₃;

R² is aryl, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, or (C₂-C₆)alkynyl, and isoptionally substituted with one to three halo, (C₁-C₄)alkyl,(C₂-C₄)alkenyl, (C₂-C₄)alkynyl, COR⁶, COOR⁶, CON(R⁶)₂, N(R⁶)₂,NR⁶CON(R⁶)₂, NR⁶CSN(R⁶)₂, OR⁶, SR⁶, CN, NO₂, or N₃;

R³ is H, halo, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, COR⁶,COOR⁶, CON(R⁶)₂, N(R⁶)₂, NR⁶CON(R⁶)₂, NR⁶CSN(R⁶)₂, OR⁶, S(O)_(p)R⁶, CN,NO₂, S(O)_(p)N(R⁶)₂ or N₃, and is optionally and independentlysubstituted with halo, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₁-C₆)alkoxy,(C₁-C₆)haloalkoxy, (C₁-C₆)hydroxyalkyl, —OH, —NH₂, NH(C₁-C₃)alkyl,N((C₁-C₃)alkyl)₂, or CN;

R⁴ is H, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, OR⁶, or CON(R⁶)₂;

each R⁵ is independently halo, (C₁-C₆)alkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, COR⁶, COOR⁶, CON(R⁶)₂, N(R⁶)₂, NR⁶CON(R⁶)₂, NR⁶CSN(R⁶)₂,OR⁶, S(O)_(p)R⁶, CN, NO₂, or N₃, and is optionally substituted withhalo, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₁-C₆)alkoxy, (C₁-C₆)haloalkoxy,(C₁-C₆)hydroxyalkyl, —OH, —NH₂, NH(C₁-C₃)alkyl, N((C₁-C₃)alkyl)₂, or CN;

each R⁶ is independently H, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, or(C₂-C₆)alkynyl;

n is 0, 1, 2, 3, 4 or 5; and

p is 0, 1 or 2.

In an embodiment of formula (III), when Z is a bond, Y₂ is CH, and R² ismethyl, then R¹ is other than furan-2-yl, 1,3-oxazol-2-yl, furan-3-yl,1,3-oxazol-5-yl, pyridine-3-yl, 1,3-thiazol-2-yl,1-methyl-1H-imidazol-5-yl, 1H-imidazol-5-yl, 3-chloro-pyridin-4-yl,3-methyl-pyridin-4-yl, 3-methoxy-pyridin-4-yl,2-methoxy-5-chloro-pyridin-3-yl, 1,3,4-oxadiazol-2-yl, and1-ethyl-1H-imidazol-5-yl.

In other embodiments of formula (III), R¹ has one substituent; Z is acarbonyl group; Z is a bond; R¹ is pyridyl, pyrazolyl, isoxazolyl ortetrazolyl; X₁ and X₂ are C; Y₂ is C; R³ is H; R⁴ is H; n is 2; and R⁵is halo; R¹ is heteroaryl; R³ and R⁴ are H; and n is 2, and R⁵ is halo.

In other aspects, pharmaceutical compositions including apharmaceutically acceptable carrier and a compound of the invention aredisclosed. The composition may further include one or more additionaltherapeutic agents, e.g., immunosuppressive agents, anti-inflammatoryagents and suitable mixtures thereof. Other additional therapeuticagents include steroids, non-steroidal anti-inflammatory agents,antihistamines, analgesics, and suitable mixtures thereof.

Compounds as disclosed herein, or a pharmaceutically acceptable salt,solvate, clathrate, or prodrug thereof, are particularly usefulinhibiting immune cell (e.g., T-cells and/or B-cells) activation (e.g.,activation in response to an antigen). In particular, these compounds ora pharmaceutically acceptable salt, solvate, clathrate, or prodrugthereof can inhibit the production of certain cytokines that regulateimmune cell activation. For example, a compound of the invention or apharmaceutically acceptable salt, solvate, clathrate, or prodrug thereofcan inhibit the production of IL-2, IL-4, IL-5, IL-13, GM-CSF, TNFα,IFN-γ or combinations thereof. Moreover, a compound of the invention ora pharmaceutically acceptable salt, solvate, clathrate, or prodrugthereof can modulate the activity of one or more ion channel involved inactivation of immune cells, such as CRAC ion channels.

A compound of the invention or a pharmaceutically acceptable salt,solvate, clathrate, or prodrug thereof is particularly useful forimmunosuppression or for treating or preventing inflammatory conditions,allergic disorders, and immune disorders.

The invention also encompasses pharmaceutical compositions comprising acompound of the invention or a pharmaceutically acceptable salt,solvate, clathrate, or prodrug thereof; and a pharmaceuticallyacceptable carrier or vehicle. These compositions may further compriseadditional agents. These compositions are useful for immunosuppressionand treating or preventing inflammatory conditions, allergic disordersand immune disorders.

The invention further encompasses methods for treating or preventinginflammatory conditions, allergic disorders, and immune disorders,comprising administering to a subject in need thereof an effectiveamount of a compound of the invention or a pharmaceutically acceptablesalt, solvate, clathrate, or prodrug thereof, or a pharmaceuticalcomposition comprising a compound of the invention or a pharmaceuticallyacceptable salt, solvate, clathrate, or prodrug thereof. These methodsmay also comprise administering to the subject an additional agentseparately or in a combination composition with the compound of theinvention or a pharmaceutically acceptable salt, solvate, clathrate, orprodrug thereof.

The invention further encompasses methods for suppressing the immunesystem of a subject, comprising administering to a subject in needthereof an effective amount of a compound of the invention or apharmaceutically acceptable salt, solvate, clathrate, or prodrugthereof, or a pharmaceutical composition comprising a compound of theinvention or a pharmaceutically acceptable salt, solvate, clathrate, orprodrug thereof. These methods may also comprise administering to thesubject an additional agent separately or in a combination compositionwith the compound of the invention or a pharmaceutically acceptablesalt, solvate, clathrate, or prodrug thereof.

The invention further encompasses methods for inhibiting immune cellactivation, including inhibiting proliferation of T cells and/or Bcells, in vivo or in vitro comprising administering to the cell aneffective amount of a compound of the invention or a pharmaceuticallyacceptable salt, solvate, clathrate, or prodrug thereof or apharmaceutical composition comprising a compound of the invention or apharmaceutically acceptable salt, solvate, clathrate, or prodrugthereof.

The invention further encompasses methods for inhibiting cytokineproduction in a cell, (e.g., IL-2, IL-4, IL-5, IL-13, GM-CSF, TNFα,and/or IFN-γ production) in vivo or in vitro comprising administering toa cell an effective amount of a compound of the invention or apharmaceutically acceptable salt, solvate, clathrate, or prodrug thereofor a pharmaceutical composition comprising a compound of the inventionor a pharmaceutically acceptable salt, solvate, clathrate, or prodrugthereof.

The invention further encompasses methods for modulating ion channelactivity (e.g., CRAC) in vivo or in vitro comprising administering aneffective amount of a compound of the invention or a pharmaceuticallyacceptable salt, solvate, clathrate, or prodrug thereof or apharmaceutical composition comprising a compound of the invention or apharmaceutically acceptable salt, solvate, clathrate, or prodrugthereof.

All of the methods of this invention may be practiced with a compound ofthe invention alone, or in combination with other agents, such as otherimmunosuppressive agents, anti-inflammatory agents, agents for thetreatment of allergic disorders or agents for the treatment of immunedisorders.

In certain embodiments of Formula (I), when Z is a bond, Y₂ is CH, X₁and X₂ are both C, and R² is methyl, then R¹ is other than furan-2-yl,1,3-oxazol-2-yl, furan-3-yl, 1,3-oxazol-5-yl, pyridine-3-yl,1,3-thiazol-2-yl, 1-methyl-1H-imidazol-5-yl, 1H-imidazol-5-yl,3-chloro-pyridin-4-yl, 3-methyl-pyridin-4-yl, 3-methoxy-pyridin-4-yl,2-methoxy-5-chloro-pyridin-3-yl, 1,3,4-oxadiazol-2-yl, and1-ethyl-1H-imidazol-5-yl; when Z is a bond, Y₂ is CH, X₁ is N, X₂ is C,and R² is methyl, then R¹— is other than 1,3-oxazol-5-yl; when Z is abond, Y₂ is CH, X₁ is C, X₂ is N and R² is methyl, then R¹— is otherthan 1-methyl-1H-imidazol-5-yl, 1,3-oxazol-5-yl, isoxazol-5-yl,1,3-thiazol-2-yl and 1H-imidazol-5-yl; and when Z is a bond, Y₂ is CH,X₁ and X₂ are both N and R² is methyl, then R¹— is other than1,3-oxazol-5-yl, 1-methyl-1H-imidazol-5-yl, 1H-imidazol-5-yl,1,3-thiazol-2-yl.

In other embodiments, the following compounds are explicitly excludedfrom formula I:

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term an “aromatic ring” or “aryl” means a monocyclicor polycyclic-aromatic ring or ring radical comprising carbon andhydrogen atoms. Examples of suitable aryl groups include, but are notlimited to, phenyl, tolyl, anthracenyl, fluorenyl, indenyl, azulenyl,and naphthyl, as well as benzo-fused carbocyclic moieties such as5,6,7,8-tetrahydronaphthyl. An aryl group can be unsubstituted orsubstituted with one or more substituents (including without limitationalkyl (preferably, lower alkyl or alkyl substituted with one or morehalo), hydroxy, alkoxy (preferably, lower alkoxy), alkylthio, cyano,halo, amino, and nitro. In certain embodiments, the aryl group is amonocyclic ring, wherein the ring comprises 6 carbon atoms.

As used herein, the term “alkyl” means a saturated straight chain orbranched non-cyclic hydrocarbon typically having from 1 to 10 carbonatoms. Representative saturated straight chain alkyls include methyl,ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyland n-decyl; while saturated branched alkyls include isopropyl,sec-butyl, isobutyl, tert-butyl, isopentyl, 2-methylbutyl,3-methylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl,2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl,2,3-dimethylbutyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl,2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl,2,2-dimethylpentyl, 2,2-dimethylhexyl, 3,3-dimtheylpentyl,3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylpentyl, 3-ethylpentyl,2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl,2-methyl-3-ethylpentyl, 2-methyl-4-ethylpentyl, 2-methyl-2-ethylhexyl,2-methyl-3-ethylhexyl, 2-methyl-4-ethylhexyl, 2,2-diethylpentyl,3,3-diethylhexyl, 2,2-diethylhexyl, 3,3-diethylhexyl and the like. Alkylgroups included in compounds of this invention may be optionallysubstituted with one or more substituents, such as amino, alkylamino,alkoxy, alkylthio, oxo, halo, acyl, nitro, hydroxyl, cyano, aryl,alkylaryl, aryloxy, arylthio, arylamino, carbocyclyl, carbocyclyloxy,carbocyclylthio, carbocyclylamino, heterocyclyl, heterocyclyloxy,heterocyclylamino, heterocyclylthio, and the like. In addition, anycarbon in the alkyl segment may be substituted with oxygen (═O), sulfur(═S), or nitrogen (═NR²³, wherein R²³ is —H, an alkyl, acetyl, oraralkyl). Lower alkyls are typically preferred for the compounds of thisinvention.

The term alkylene refers to an alkyl group that has two points ofattachment to two moieties (e.g., {—CH₂—}, —{CH₂CH₂—},

etc., wherein the brackets indicate the points of attachment). Alkylenegroups may be substituted or unsubstituted, as with an alkyl group.

An aralkyl group refers to an aryl group that is attached to anothermoiety via an alkylene linker. Aralkyl groups can be substituted orunsubstituted, as with an aryl group and/or alkyl group.

The term “alkoxy,” as used herein, refers to an alkyl group that islinked to another moiety though an oxygen atom. Alkoxy groups can besubstituted or unsubstituted, as with an alkyl group.

The term “alkoxyalkoxy,” as used herein, refers to an alkoxy group inwhich the alkyl portion is substituted with another alkoxy group.

The term “alkyl sulfanyl,” as used herein, refers to an alkyl group thatis linked to another moiety though a divalent sulfur atom. Alkylsulfanyl groups can be substituted or unsubstituted, as with an alkylgroup.

The term “alkylamino,” as used herein, refers to an amino group in whichone hydrogen atom attached to the nitrogen has been replaced by an alkylgroup. The term “dialkylamino,” as used herein, refers to an amino groupin which two hydrogen atoms attached to the nitrogen have been replacedby alkyl groups, in which the alkyl groups can be the same or different.Alkylamino groups and dialkylamino groups can be substituted orunsubstituted, as with an alkyl group.

As used herein, the term “alkenyl” means a straight chain or branched,hydrocarbon radical typically having from 2 to 10 carbon atoms andhaving at least one carbon-carbon double bond. Representative straightchain and branched alkenyls include vinyl, allyl, 1-butenyl, 2-butenyl,isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl,1-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, 1-hexenyl, 2-hexenyl,3-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 1-octenyl, 2-octenyl,3-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 2-decenyl,3-decenyl and the like. Alkenyl groups can be substituted orunsubstituted, as with alkyl groups.

As used herein, the term “alkynyl” means a straight chain or branched,hydrocarbonon radical typically having from 2 to 10 carbon atoms andhaving at least one carbon-carbon triple bond. Representative straightchain and branched alkynyls include acetylenyl, propynyl, 1-butynyl,2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl,4-pentynyl,-1-hexynyl, 2-hexynyl, 5-hexynyl, 1-heptynyl, 2-heptynyl,6-heptynyl, 1-octynyl, 2-octynyl, 7-octynyl, 1-nonynyl, 2-nonynyl,8-nonynyl, 1-decynyl, 2-decynyl, 9-decynyl and the like. Alkynyl groupscan be substituted or unsubstituted.

As used herein, the term “cycloalkyl” means a saturated, mono- orpolycyclic alkyl radical typically having from 3 to 10 carbon atoms.Representative cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, adamantlyl,decahydronaphthyl, octahydropentalene, bicyclo[1,1,1]pentanyl, and thelike. Cycloalkyl groups can be substituted or unsubstituted, as withalkyl groups.

As used herein, the term “cycloalkenyl” means a cyclic non-aromaticalkenyl radical having at least one carbon-carbon double bond in thecyclic system and typically having from 5 to 10 carbon atoms.Representative cycloalkenyls include cyclopentenyl, cyclopentadienyl,cyclohexenyl, cyclohexadienyl, cycloheptenyl, cycloheptadienyl,cycloheptatrienyl, cyclooctenyl, cyclooctadienyl, cyclooctatrienyl,cyclooctatetraenyl, cyclononenyl, cyclononadienyl, cyclodecenyl,cyclodecadienyl and the like. Cycloalkenyl groups can be substituted orunsubstituted, as with alkyl groups.

As used herein, the term “heterocycle” or “heterocyclyl” means amonocyclic or polycyclic heterocyclic ring (typically having 3- to14-members) that is either a saturated ring or an unsaturatednon-aromatic ring. A 3-membered heterocycle can contain up to 3heteroatoms, and a 4- to 14-membered heterocycle can contain from 1 toabout 8 heteroatoms. Each heteroatom is independently selected fromnitrogen, which can be quaternized; oxygen; and sulfur, includingsulfoxide and sulfone. The heterocycle may be attached via anyheteroatom or carbon atom. Representative heterocycles includemorpholinyl, thiomorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl,piperazinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl,tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyrindinyl,tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, andthe like. A heteroatom may be substituted with a protecting group knownto those of ordinary skill in the art, for example, the hydrogen on anitrogen may be substituted with a tert-butoxycarbonyl group.Furthermore, the heterocyclyl may be optionally substituted with one ormore substituents (including without limitation a halogen atom, an alkylradical, or aryl radical). Only stable isomers of such substitutedheterocyclic groups are contemplated in this definition.

As used herein, the term “heteroaromatic” or “heteroaryl” means amonocyclic or polycyclic heteroaromatic ring (or radical thereof)comprising carbon atom ring members and one or more heteroatom ringmembers (such as, for example, oxygen, sulfur or nitrogen). Typically,the heteroaromatic ring has from 5 to about 14 ring members in which atleast 1 ring member is a heteroatom selected from oxygen, sulfur, andnitrogen. In another embodiment, the heteroaromatic ring is a 5 or 6membered ring and may contain from 1 to about 4 heteroatoms. In anotherembodiment, the heteroaromatic ring system has a 7 to 14 ring membersand may contain from 1 to about 7 heteroatoms. Representativeheteroaryls include pyridyl, furyl, thienyl, pyrrolyl, oxazolyl,imidazolyl, indolizinyl, thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl,pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, triazolyl, pyridinyl,thiadiazolyl, pyrazinyl, quinolyl, isoquniolyl, indazolyl, benzoxazolyl,benzofuryl, benzothiazolyl, indolizinyl, imidazopyridinyl, isothiazolyl,tetrazolyl, benzimidazolyl, benzoxazolyl, benzothiazolyl,benzothiadiazolyl, benzoxadiazolyl, indolyl, tetrahydroindolyl,azaindolyl, imidazopyridyl, qunizaolinyl, purinyl,pyrrolo[2,3]pyrimidyl, pyrazolo[3,4]pyrimidyl, benzo(b)thienyl, and thelike. These heteroaryl groups may be optionally substituted with one ormore substituents.

A heteroaralkyl group refers to a heteroaryl group that is attached toanother moiety via an alkylene linker. Heteroaralkyl groups can besubstituted or unsubstituted.

As used herein, the term “halogen” or “halo” means —F, —Cl, —Br, or —I.

As used herein, the term “haloalkyl” means an alkyl group in which oneor more —H is replaced with a halo group. Examples of haloalkyl groupsinclude —CF₃, —CHF₂, —CCl₃, —CH₂CH₂Br, —CH₂CH(CH₂CH₂Br)CH₃, —CHICH₃, andthe like.

As used herein, the term “haloalkoxy” means an alkoxy group in which oneor more —H is replaced with a halo group. Examples of haloalkoxy groupsinclude —OCF₃ and —OCHF₂.

As used herein, the term “linker” means a diradical having from 1-6atoms connected together so as to form an uninterrupted array or seriesof atoms, and which covalently connects two other moieties. For example,a linker of the compounds described herein having a specified number ofatoms in contiguous connectivity has at least that number of atomsconnected together so as to form an uninterrupted chain, but may alsoinclude additional atoms that are not so connected (e.g., branches oratoms contained within a ring system). The atoms of the linker may beconnected by saturated or unsaturated covalent bonds. Linkers include,but are not limited to, alkylidene, alkenylidene, alkynylidene andcycloalkylidene (such as lower alkylidene, cycloalkylidene,alkylycloalkylidene and alkyl-substituted alkylidene) linkers whereinone or more (e.g., between 1 and 3, (e.g., 1 or 2)) carbon atoms may beoptionally replaced with O, S, or N and wherein two or more (e.g., 2-3(e.g., 2 or 3)) adjacent atoms may be optionally linked together to forma carbocyclic or heterocyclic moiety within the linker (which may bemonocyclic, polycyclic and/or fused, and which may be saturated,unsaturated, or aromatic). Examples of specific linkers useful in thecompounds of the invention include (without limitation) diradicals ofalkyl, alkenyl, alynyl, alkoxy, alkoxyalkyl, alkylaminoalkyl,cycloalkyl, alkylcycloalkyl, and alkyl-substituted alkylcycloalkyl(wherein one or more carbon atoms in any of these linkers may beoptionally replaced with O, S, or N).

As used herein, the terms “subject,” “patient,” and “animal”, are usedinterchangeably and include, but are not limited to, a cow, monkey,horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit,guinea pig, or human. The preferred subject, patient, or animal is ahuman.

As used herein, the term “lower” refers to a group having up to fourcarbon atoms. For example, a “lower alkyl” refers to an alkyl radicalhaving from 1 to 4 carbon atoms, and a “lower alkenyl” or “loweralkynyl” refers to an alkenyl or alkynyl radical having from 2 to 4carbon atoms, respectively. A lower alkoxy or a lower alkyl sulfanylrefers to an alkoxy or an alkyl sulfanyl having from 1 to 4 carbonatoms. Lower substituents are typically preferred.

Where a particular substituent, such as an alkyl substituent, occursmultiple times in a given structure or moiety, the identity of thesubstituent is independent in each case and may be the same as ordifferent from other occurrences of that substituent in the structure ormoiety. Furthermore, individual substituents in the specific embodimentsand exemplary compounds of this invention are preferred in combinationwith other such substituents in the compounds of this invention, even ifsuch individual substituents are not expressly noted as being preferredor not expressly shown in combination with other substituents.

The compounds of the invention are defined herein by their chemicalstructures and/or chemical names. Where a compound is referred to byboth a chemical structure and a chemical name, and the chemicalstructure and chemical name conflict, the chemical structure isdeterminative of the compound's identity.

Suitable substituents for an alkyl, alkoxy, alkyl sulfanyl, alkylamino,dialkylamino, alkylene, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,heterocyclyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl groupsinclude any substituent that will form a stable compound of theinvention. Examples of substituents for an alkyl, alkoxy, alkylsulfanyl,alkylamino, dialkylamino, alkylene, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, heterocyclyl, aryl, aralkyl, heteroaryl, andheteroarylalkyl include an alkyl, alkoxy, alkyl sulfanyl, alkylamino,dialkylamino, an alkenyl, an alkynyl, an cycloalkyl, an cycloalkenyl, anheterocyclyl, an aryl, an heteroaryl, an aralkyl, an heteroaralkyl, ahaloalkyl, —C(O)NR₁₃R₁₄, —NR₁₅C(O)R₁₆, halo, —OR₁₅, cyano, nitro,haloalkoxy, —C(O)R₁₅, —NR₁₃R₁₄, —SR₁₅, —C(O)OR₁₅, —OC(O)R₁₅,—NR₁₅C(O)NR₁₃R₁₄, —OC(O)NR₁₃R₁₄, —NR₁₅C(O)OR₁₆, —S(O)_(p)R₁₅, or—S(O)_(p)NR₁₃R₁₄, wherein R₁₃ and R₁₄, for each occurrence are,independently, H, an optionally substituted alkyl, an optionallysubstituted alkenyl, an optionally substituted alkynyl, an optionallysubstituted cycloalkyl, an optionally substituted cycloalkenyl, anoptionally substituted heterocyclyl, an optionally substituted aryl, anoptionally substituted heteroaryl, an optionally substituted aralkyl, oran optionally substituted heteroaralkyl; or R₁₃ and R₁₄ taken togetherwith the nitrogen to which they are attached form optionally substitutedheterocyclyl or optionally substituted heteroaryl; and R₁₅ and R₁₆ foreach occurrence are, independently, H, an optionally substituted alkyl,an optionally substituted alkenyl, an optionally substituted alkynyl, anoptionally substituted cycloalkyl, an optionally substitutedcycloalkenyl, an optionally substituted heterocyclyl, an optionallysubstituted aryl, an optionally substituted heteroaryl, an optionallysubstituted aralkyl, or an optionally substituted heteroaralkyl.

In addition, alkyl, cycloalkyl, alkylene, heterocyclyl, and anysaturated portion of a alkenyl, cycloalkenyl, alkynyl, aralkyl, orheteroaralkyl group, may also be substituted with ═O, ═S, or ═N—R₁₅.

When a heterocyclyl, heteroaryl, or heteroaralkyl group contains anitrogen atom, it may be substituted or unsubstituted. When a nitrogenatom in the aromatic ring of a heteroaryl group has a substituent thenitrogen may be a quaternary nitrogen.

Choices and combinations of substituents and variables envisioned bythis invention are only those that result in the formation of stablecompounds. The term “stable”, as used herein, refers to compounds whichpossess stability sufficient to allow manufacture and which maintainsthe integrity of the compound for a sufficient period of time to beuseful for the purposes detailed herein (e.g., therapeutic orprophylactic administration to a subject). Typically, such compounds arestable at a temperature of 40° C. or less, in the absence of excessivemoisture, for at least one week. Such choices and combinations will beapparent to those of ordinary skill in the art and may be determinedwithout undue experimentation.

Unless indicated otherwise, the compounds of the invention containingreactive functional groups (such as, without limitation, carboxy,hydroxy, and amino moieties) also include protected derivatives thereof.“Protected derivatives” are those compounds in which a reactive site orsites are blocked with one ore more protecting groups. Suitableprotecting groups for carboxy moieties include benzyl, tert-butyl, andthe like. Suitable protecting groups for amino and amido groups includeacetyl, tert-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitableprotecting groups for hydroxy include benzyl and the like. Othersuitable protecting groups are well known to those of ordinary skill inthe art and include those found in T. W. Greene, PROTECTING GROUPS INORGANIC SYNTHESIS, John Wiley & Sons, Inc. 1981, the entire teachings ofwhich are incorporated herein by reference.

As used herein, the term “compound(s) of this invention” and similarterms refers to a compound of formula I or a pharmaceutically acceptablesalt, solvate, clathrate, or prodrug thereof and also include protectedderivatives thereof.

As used herein and unless otherwise indicated, the term “prodrug” meansa derivative of a compound that can hydrolyze, oxidize, or otherwisereact under biological conditions (in vitro or in vivo) to provide acompound of this invention. Prodrugs may only become active upon suchreaction under biological conditions, but they may have activity intheir unreacted forms. Examples of prodrugs contemplated in thisinvention include, but are not limited to, analogs or derivatives ofcompounds of the invention that comprise biohydrolyzable moieties suchas biohydrolyzable amides, biohydrolyzable esters, biohydrolyzablecarbamates, biohydrolyzable carbonates, biohydrolyzable ureides, andbiohydrolyzable phosphate analogues. Other examples of prodrugs includederivatives of compounds of the invention that include —NO, —NO₂, —ONO,or —ONO₂ moieties. Prodrugs can typically be prepared using well-knownmethods, such as those described by BURGER'S MEDICINAL CHEMISTRY ANDDRUG DISCOVERY (1995) 172-178, 949-982 (Manfred E. Wolff ed., 5th ed),the entire teachings of which are incorporated herein by reference.

As used herein and unless otherwise indicated, the terms“biohydrolyzable amide”, “biohydrolyzable ester”, “biohydrolyzablecarbamate”, “biohydrolyzable carbonate”, “biohydrolyzable ureide” and“biohydrolyzable phosphate analogue” mean an amide, ester, carbamate,carbonate, ureide, or phosphate analogue, respectively, that either: 1)does not destroy the biological activity of the compound and confersupon that compound advantageous properties in vivo, such as uptake,duration of action, or onset of action; or 2) is itself biologicallyinactive but is converted in vivo to a biologically active compound.Examples of biohydrolyzable amides include, but are not limited to,lower alkyl amides, α-amino acid amides, alkoxyacyl amides, andalkylaminoalkylcarbonyl amides. Examples of biohydrolyzable estersinclude, but are not limited to, lower alkyl esters, alkoxyacyloxyesters, alkyl acylamino alkyl esters, and choline esters. Examples ofbiohydrolyzable carbamates include, but are not limited to, loweralkylamines, substituted ethylenediamines, amino acids,hydroxyalkylamines, heterocyclic and heteroaromatic amines, andpolyether amines.

As used herein, the term “pharmaceutically acceptable salt,” is a saltformed from an acid and a basic group of one of the compounds of theinvention. Illustrative salts include, but are not limited, to sulfate,citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate,and pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts.The term “pharmaceutically acceptable salt” also refers to a saltprepared from a compound of the invention having an acidic functionalgroup, such as a carboxylic acid functional group, and apharmaceutically acceptable inorganic or organic base. Suitable basesinclude, but are not limited to, hydroxides of alkali metals such assodium, potassium, and lithium; hydroxides of alkaline earth metal suchas calcium and magnesium; hydroxides of other metals, such as aluminumand zinc; ammonia, and organic amines, such as unsubstituted orhydroxy-substituted mono-, di-, or trialkylamines; dicyclohexylamine;tributyl amine; pyridine; N-methyl,N-ethylamine; diethylamine;triethylamine; mono-, bis-, or tris-(2-hydroxy-lower alkyl amines), suchas mono-, bis-, or tris-(2-hydroxyethyl)-amine,2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine,N,N,-di-lower alkyl-N-(hydroxy lower alkyl)-amines, such asN,N-dimethyl-N-(2-hydroxyethyl)-amine, or tri-(2-hydroxyethyl)amine;N-methyl-D-glucamine; and amino acids such as arginine, lysine, and thelike. The term “pharmaceutically acceptable salt” also refers to a saltprepared from a compound of the invention having a basic functionalgroup, such as an amino functional group, and a pharmaceuticallyacceptable inorganic or organic acid. Suitable acids include, but arenot limited to, hydrogen sulfate, citric acid, acetic acid, oxalic acid,hydrochloric acid, hydrogen bromide, hydrogen iodide, nitric acid,phosphoric acid, isonicotinic acid, lactic acid, salicylic acid,tartaric acid, ascorbic acid, succinic acid, maleic acid, besylic acid,fumaric acid, gluconic acid, glucaronic acid, saccharic acid, formicacid, benzoic acid, glutamic acid, methanesulfonic acid, ethanesulfonicacid, benzenesulfonic acid, and p-toluenesulfonic acid.

When a disclosed compound is named or depicted by structure, it is to beunderstood that solvates (e.g., hydrates) of the compound or itspharmaceutically acceptable salts are also included. “Solvates” refer tocrystalline forms wherein solvent molecules are incorporated into thecrystal lattice during crystallization. Solvate may include water ornonaqueous solvents such as ethanol, isopropanol, DMSO, acetic acid,ethanolamine, and EtOAc. Solvates, wherein water is the solvent moleculeincorporated into the crystal lattice, are typically referred to as“hydrates”. Hydrates include a stoichiometric or non-stoichiometricamount of water bound by non-covalent intermolecular forces.

When a disclosed compound is named or depicted by structure, it is to beunderstood that the compound, including solvates thereof, may exist incrystalline forms, non-crystalline forms or a mixture thereof. Thecompounds or solvates may also exhibit polymorphism (i.e., the capacityto occur in different crystalline forms). These different crystallineforms are typically known as “polymorphs.” It is to be understood thatwhen named or depicted by structure, the disclosed compounds andsolvates (e.g., hydrates) also include all polymorphs thereof. As usedherein, the term “polymorph” means solid crystalline forms of a compoundof the present invention or complex thereof. Different polymorphs of thesame compound can exhibit different physical, chemical and/orspectroscopic properties. Different physical properties include, but arenot limited to stability (e.g., to heat or light), compressibility anddensity (important in formulation and product manufacturing), anddissolution rates (which can affect bioavailability). Differences instability can result from changes in chemical reactivity (e.g.,differential oxidation, such that a dosage form discolors more rapidlywhen comprised of one polymorph than when comprised of anotherpolymorph) or mechanical characteristics (e.g., tablets crumble onstorage as a kinetically favored polymorph converts to thermodynamicallymore stable polymorph) or both (e.g., tablets of one polymorph are moresusceptible to breakdown at high humidity). Different physicalproperties of polymorphs can affect their processing. For example, onepolymorph might be more likely to form solvates or might be moredifficult to filter or wash free of impurities than another due to, forexample, the shape or size distribution of particles of it. In addition,one polymorph may spontaneously convert to another polymorph undercertain conditions.

When a disclosed compound is named or depicted by structure, it is to beunderstood that clathrates (“inclusion compounds”) of the compound orits pharmaceutically acceptable salts, solvates or polymorphs are alsoincluded. As used herein, the term “clathrate” means a compound of thepresent invention or a salt thereof in the form of a crystal latticethat contains spaces (e.g., channels) that have a guest molecule (e.g.,a solvent or water) trapped within.

As used herein, the term “asthma” means a pulmonary disease, disorder orcondition characterized by reversible airway obstruction, airwayinflammation, and increased airway responsiveness to a variety ofstimuli.

“Immunosuppression” refers to impairment of any component of the immunesystem resulting in decreased immune function. This impairment may bemeasured by any conventional means including whole blood assays oflymphocyte function, detection of lymphocyte proliferation andassessment of the expression of T cell surface antigens. The antisheepred blood cell (SRBC) primary (IgM) antibody response assay (usuallyreferred to as the plaque assay) is one specific method. This and othermethods are described in Luster, M. I., Portier, C., Pait, D. G., White,K. L., Jr., Gennings, C., Munson, A. E., and Rosenthal, G. J. (1992).“Risk Assessment in Immunotoxicology I: Sensitivity and Predictabilityof Immune Tests.” Fundam. Appl. Toxicol., 18, 200-210. Measuring theimmune response to a T-cell dependent immunogen is another particularlyuseful assay (Dean, J. H., House, R. V., and Luster, M. I. (2001).“Immunotoxicology: Effects of, and Responses to, Drugs and Chemicals.”In PRINCIPLES AND METHODS OF TOXICOLOGY: FOURTH EDITION (A. W. Hayes,Ed.), pp. 1415-1450, Taylor & Francis, Philadelphia, Pa.).

The compounds of this invention can be used to treat subjects withimmune disorders. As used herein, the term “immune disorder” and liketerms means a disease, disorder or condition caused by the immune systemof an animal, including autoimmune disorders. Immune disorders includethose diseases, disorders or conditions that have an immune componentand those that are substantially or entirely immune system-mediated.Autoimmune disorders are those wherein the animal's own immune systemmistakenly attacks itself, thereby targeting the cells, tissues, and/ororgans of the animal's own body. For example, the autoimmune reaction isdirected against the nervous system in multiple sclerosis and the gut inCrohn's disease. In other autoimmune disorders such as systemic lupuserythematosus (lupus), affected tissues and organs may vary amongindividuals with the same disease. One person with lupus may haveaffected skin and joints whereas another may have affected skin, kidney,and lungs. Ultimately, damage to certain tissues by the immune systemmay be permanent, as with destruction of insulin-producing cells of thepancreas in Type 1 diabetes mellitus. Specific autoimmune disorders thatmay be ameliorated using the compounds and methods of this inventioninclude without limitation, autoimmune disorders of the nervous system(e.g., multiple sclerosis, myasthenia gravis, autoimmune neuropathiessuch as Guillain-Barré, and autoimmune uveitis), autoimmune disorders ofthe blood (e.g., autoimmune hemolytic anemia, pernicious anemia, andautoimmune thrombocytopenia), autoimmune disorders of the blood vessels(e.g., temporal arteritis, anti-phospholipid syndrome, vasculitides suchas Wegener's granulomatosis, and Behcet's disease), autoimmune disordersof the skin (e.g., psoriasis, dermatitis herpetiformis, pemphigusvulgaris, and vitiligo), autoimmune disorders of the gastrointestinalsystem (e.g., Crohn's disease, ulcerative colitis, primary biliarycirrhosis, and autoimmune hepatitis), autoimmune disorders of theendocrine glands (e.g., Type 1 or immune-mediated diabetes mellitus,Grave's disease. Hashimoto's thyroiditis, autoimmune oophoritis andorchitis, and autoimmune disorder of the adrenal gland); and autoimmunedisorders of multiple organs (including connective tissue andmusculoskeletal system diseases) (e.g., rheumatoid arthritis, systemiclupus erythematosus, scleroderma, polymyositis, dermatomyositis,spondyloarthropathies such as ankylosing spondylitis, and Sjogren'ssyndrome). In addition, other immune system mediated diseases, such asgraft-versus-host disease and allergic disorders, are also included inthe definition of immune disorders herein. Because a number of immunedisorders are caused by inflammation, there is some overlap betweendisorders that are considered immune disorders and inflammatorydisorders. For the purpose of this invention, in the case of such anoverlapping disorder, it may be considered either an immune disorder oran inflammatory disorder. “Treatment of an immune disorder” hereinrefers to administering a compound or a composition of the invention toa subject, who has an immune disorder, a symptom of such a disease or apredisposition towards such a disease, with the purpose to cure,relieve, alter, affect, or prevent the autoimmune disorder, the symptomof it, or the predisposition towards it.

As used herein, the term “allergic disorder” means a disease, conditionor disorder associated with an allergic response against normallyinnocuous substances. These substances may be found in the environment(such as indoor air pollutants and aeroallergens) or they may benon-environmental (such as those causing dermatological or foodallergies). Allergens can enter the body through a number of routes,including by inhalation, ingestion, contact with the skin or injection(including by insect sting). Many allergic disorders are linked toatopy, a predisposition to generate the allergic antibody IgE. BecauseIgE is able to sensitize mast cells anywhere in the body, atopicindividuals often express disease in more than one organ. For thepurpose of this invention, allergic disorders include anyhypersensitivity that occurs upon re-exposure to the sensitizingallergen, which in turn causes the release of inflammatory mediators.Allergic disorders include without limitation, allergic rhinitis (e.g.,hay fever), sinusitis, rhinosinusitis, chronic or recurrent otitismedia, drug reactions, insect sting reactions, latex reactions,conjunctivitis, urticaria, anaphylaxis and anaphylactoid reactions,atopic dermatitis, asthma, and food allergies.

The compounds of this invention can be used to prevent or to treatsubjects with inflammatory disorders. As used herein, an “inflammatorydisorder” means a disease, disorder or condition characterized byinflammation of body tissue or having an inflammatory component. Theseinclude local inflammatory responses and systemic inflammation. Examplesof such inflammatory disorders include: transplant rejection, includingskin graft rejection; chronic inflammatory disorders of the joints,including arthritis, rheumatoid arthritis, osteoarthritis and bonediseases associated with increased bone resorption; inflammatory boweldiseases such as ileitis, ulcerative colitis, Barrett's syndrome, andCrohn's disease; inflammatory lung disorders such as asthma, adultrespiratory distress syndrome, and chronic obstructive airway disease;inflammatory disorders of the eye including corneal dystrophy, trachoma,onchocerciasis, uveitis, sympathetic ophthalmitis and endophthalmitis;chronic inflammatory disorders of the gums, including gingivitis andperiodontitis; tuberculosis; leprosy; inflammatory diseases of thekidney including uremic complications, glomerulonephritis and nephrosis;inflammatory disorders of the skin including sclerodermatitis, psoriasisand eczema; inflammatory diseases of the central nervous system,including chronic demyelinating diseases of the nervous system, multiplesclerosis, AIDS-related neurodegeneration and Alzheimer's disease,infectious meningitis, encephalomyelitis, Parkinson's disease,Huntington's disease, amyotrophic lateral sclerosis and viral orautoimmune encephalitis; autoimmune disorders, immune-complexvasculitis, systemic lupus and erythematodes; systemic lupuserythematosus (SLE); and inflammatory diseases of the heart such ascardiomyopathy, ischemic heart disease hypercholesterolemia,atherosclerosis); as well as various other diseases with significantinflammatory components, including preeclampsia; chronic liver failure,brain and spinal cord trauma, cancer). There may also be a systemicinflammation of the body, exemplified by gram-positive or gram negativeshock, hemorrhagic or anaphylactic shock, or shock induced by cancerchemotherapy in response to pro-inflammatory cytokines, e.g., shockassociated with pro-inflammatory cytokines. Such shock can be induced,e.g., by a chemotherapeutic agent used in cancer chemotherapy.“Treatment of an inflammatory disorder” herein refers to administering acompound or a composition of the invention to a subject, who has aninflammatory disorder, a symptom of such a disorder or a predispositiontowards such a disorder, with the purpose to cure, relieve, alter,affect, or prevent the inflammatory disorder, the symptom of it, or thepredisposition towards it.

An “effective amount” is the quantity of compound in which a beneficialoutcome is achieved when the compound is administered to a subject oralternatively, the quantity of compound that possess a desired activityin vivo or in vitro. In the case of inflammatory disorders andautoimmune disorders, a beneficial clinical outcome includes reductionin the extent or severity of the symptoms associated with the disease ordisorder and/or an increase in the longevity and/or quality of life ofthe subject compared with the absence of the treatment. The preciseamount of compound administered to a subject will depend on the type andseverity of the disease or condition and on the characteristics of thesubject, such as general health, age, sex, body weight and tolerance todrugs. It will also depend on the degree, severity and type ofinflammatory disorder or autoimmune disorder or the degree ofimmunosuppression sought. The skilled artisan will be able to determineappropriate dosages depending on these and other factors. Effectiveamounts of the disclosed compounds typically range between about 1 mg/m²per day and about 10 grams/m² per day, and preferably between 10 mg/m²per day and about 1 gram/m².

The compounds of the invention may contain one or more chiral centersand/or double bonds and, therefore, exist as stereoisomers, such asdouble-bond isomers (i.e., geometric isomers), enantiomers, ordiastereomers. According to this invention, the chemical structuresdepicted herein, including the compounds of this invention, encompassall of the corresponding compounds' enantiomers and stereoisomers, thatis, both the stereomerically pure form (e.g., geometrically pure,enantiomerically pure, or diastereomerically pure) and enantiomeric,diastereomeric, and geometric isomeric mixtures. In some cases, oneenantiomer, diastereomer, or geometric isomer will possess superioractivity or an improved toxicity or kinetic profile compared to others.In those cases, such enantiomers, diastereomers, and geometric isomersof a compound of this invention are preferred.

The term “inhibit production of IL-2” and like terms means inhibitingIL-2 synthesis (e.g., by inhibiting transcription (mRNA expression), ortranslation (protein expression)) and/or inhibiting IL-2 secretion in acell that has the ability to produce and/or secrete IL-2 (e.g., Tlymphocyte). Likewise, the term “inhibiting production of IL-4, IL-5,IL-13, GM-CSF, TNFα or IFN-γ means inhibiting the synthesis (e.g., byinhibiting transcription, or translation) and/or inhibiting thesecretion in a cell that has the ability to produce and/or secrete thesecytokines.

As used herein, a racemic mixture means about 50% of one enantiomer andabout 50% of is corresponding enantiomer relative to all chiral centersin the molecule. The invention encompasses all enantiomerically-pure,enantiomerically-enriched, diastereomerically pure, diastereomericallyenriched, and racemic mixtures of the compounds of the invention.

Enantiomeric and diastereomeric mixtures can typically be resolved intotheir component enantiomers or stereoisomers by well known methods, suchas chiral-phase gas chromatography, chiral-phase high performance liquidchromatography, crystallizing the compound as a chiral salt complex, orcrystallizing the compound in a chiral solvent. Enantiomers anddiastereomers can also be obtained from diastereomerically- orenantiomerically-pure intermediates, reagents, and catalysts bywell-known asymmetric synthetic methods.

When administered to a patient, e.g., to a non-human animal forveterinary use or for improvement of livestock, or to a human forclinical use, the compounds of the invention are typically administeredin isolated form or as the isolated form in a pharmaceuticalcomposition. As used herein, “isolated” means that the compounds of theinvention are separated from other components of either (a) a naturalsource, such as a plant or cell, preferably bacterial culture, or (b) asynthetic organic chemical reaction mixture. Preferably, viaconventional techniques, the compounds of the invention are purified. Asused herein, “purified” means that when isolated, the isolate containsat least 95%, preferably at least 98%, of a single compound of theinvention by weight of the isolate.

Only those choices and combinations of substituents that result in astable structure are contemplated. Such choices and combinations will beapparent to those of ordinary skill in the art and may be determinedwithout undue experimentation.

The invention can be understood more fully by reference to the followingdetailed description and illustrative examples, which are intended toexemplify non-limiting embodiments of the invention.

SPECIFIC EMBODIMENTS

The invention relates to compounds and pharmaceutical compositions thatare particularly useful for immunosuppression or to treat or preventinflammatory conditions, immune disorders, and allergic disorders.Embodiments of the invention include those compounds describedhereinabove in the Summary, e.g., those of formulae (I), (II) and (III).

All of the features, specific embodiments and particular substituentsdisclosed herein may be combined in any combination. Each feature,embodiment or substituent disclosed in this specification may bereplaced by an alternative feature, embodiment or substituent servingthe same, equivalent, or similar purpose. In the case of chemicalcompounds, specific values for variables (e.g., values shown in theexemplary compounds disclosed herein) in any chemical formula disclosedherein can be combined in any combination resulting in a stablestructure. Furthermore, specific values (whether preferred or not) forsubstituents in one type of chemical structure may be combined withvalues for other substituents (whether preferred or not) in the same ordifferent type of chemical structure. Thus, unless expressly statedotherwise, each feature, embodiment or substituent disclosed is only anexample of a generic series of equivalent or similar features,embodiments or substituents.

Exemplary Compounds

Exemplary compounds of the invention, that have been made in accordancewith the descriptions in the examples below, are depicted in Table 1below.

TABLE 1

1

2

3

8

6

7

A

B

C

D

5

4

E

F

G

H

9

10

11

I

J

K

L

41

42

M

N

15

16

O

P

Q

R

S

17

12

13

T

A1

A2

A3

A4

A5

A6

A7

A8

A9

A10

A11

A12

14

Mechanism of Action

Activation of T-lymphocytes in response to an antigen is dependent oncalcium ion oscillations. Calcium ion oscillations in T-lymphocytes aretriggered through stimulation of the T-cell antigen receptor, andinvolve calcium ion influx through the stored-operatedCa²⁺-release-activated Ca²⁺ (CRAC) channel. Although a detailedelectrophysiological profile of the channel exists, the molecularstructure of the CRAC ion channel had not been identified till therecent identification of the pore-forming unit, named Orai1/CRACM1 (Vig,Science (2006), 312:1220-3, Feske, Nature (2006), 441:179-85). Thus,inhibition of CRAC ion channels can be measured by measuring inhibitionof the I_(CRAC) current. Calcium ion oscillations in T-cells have beenimplicated in the activation of several transcription factors (e.g.,NFAT, Oct/Oap and NFκB) which are critical for T-cell activation (Lewis,Biochemical Society Transactions (2003), 31:925-929, the entireteachings of which are incorporated herein by reference). Withoutwishing to be bound by any theory, it is believed that because thecompounds of the invention inhibit the activity of CRAC ion channels,they inhibit immune cell activation.

Methods of Treatment and Prevention

A effective amount of a compound of the invention or a pharmaceuticallyacceptable salt, solvate, clathrate, and prodrug thereof, or apharmaceutical composition comprising a compound of the invention, or apharmaceutically acceptable salt, solvate, clathrate, and prodrugthereof, is administered to a patient in need of immunosuppression or inneed of treatment or prevention of an inflammatory condition, an immunedisorder, or an allergic disorder. Such patients may be treatment naïveor may experience partial or no response to conventional therapies.

Responsiveness of a particular inflammatory condition, immune disorder,or allergic disorder in a subject can be measured directly (e.g.,measuring blood levels of inflammatory cytokines (such as IL-2, IL-4,IL-5, IL-13, GM-CSF, TNFα, IFN-γ and the like) after administration of acompound of this invention), or can be inferred based on anunderstanding of disease etiology and progression. The compounds of theinvention, or pharmaceutically acceptable salts, solvates, clathrates,and prodrugs thereof can be assayed in vitro or in vivo, for the desiredtherapeutic or prophylactic activity, prior to use in humans. Forexample, known animal models of inflammatory conditions, immunedisorders, or allergic disorders can be used to demonstrate the safetyand efficacy of compounds of this invention.

Pharmaceutical Compositions and Dosage Forms

Pharmaceutical compositions and dosage forms of the invention compriseone or more active ingredients in relative amounts and formulated insuch a way that a given pharmaceutical composition or dosage form can beused for immunosuppression or to treat or prevent inflammatoryconditions, immune disorders, and allergic disorders. Preferredpharmaceutical compositions and dosage forms comprise a compound of theinvention, or a pharmaceutically acceptable prodrug, salt, solvate, orclathrate thereof, optionally in combination with one or more additionalactive agents.

Single unit dosage forms of the invention are suitable for oral, mucosal(e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g.,subcutaneous, intravenous, bolus injection, intramuscular, orintraarterial), or transdermal administration to a patient. Examples ofdosage forms include, but are not limited to: tablets; caplets;capsules, such as soft elastic gelatin capsules; cachets; troches;lozenges; dispersions; suppositories; ointments; cataplasms (poultices);pastes; powders; dressings; creams; plasters; solutions; patches;aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage formssuitable for oral or mucosal administration to a patient, includingsuspensions (e.g., aqueous or non-aqueous liquid suspensions,oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions,and elixirs; liquid dosage forms suitable for parenteral administrationto a patient; and sterile solids (e.g., crystalline or amorphous solids)that can be reconstituted to provide liquid dosage forms suitable forparenteral administration to a patient.

The composition, shape, and type of dosage forms of the invention willtypically vary depending on their use. For example, a dosage formsuitable for mucosal administration may contain a smaller amount ofactive ingredient(s) than an oral dosage form used to treat the sameindication. This aspect of the invention will be readily apparent tothose skilled in the art. See, e.g., Remington's Pharmaceutical Sciences(1990) 18th ed., Mack Publishing, Easton Pa.

Typical pharmaceutical compositions and dosage forms comprise one ormore excipients. Suitable excipients are well known to those skilled inthe art of pharmacy, and non-limiting examples of suitable excipientsare provided herein. Whether a particular excipient is suitable forincorporation into a pharmaceutical composition or dosage form dependson a variety of factors well known in the art including, but not limitedto, the way in which the dosage form will be administered to a patient.For example, oral dosage forms such as tablets may contain excipientsnot suited for use in parenteral dosage forms.

The suitability of a particular excipient may also depend on thespecific active ingredients in the dosage form. For example, thedecomposition of some active ingredients can be accelerated by someexcipients such as lactose, or when exposed to water. Active ingredientsthat comprise primary or secondary amines (e.g., N-desmethylvenlafaxineand N,N-didesmethylvenlafaxine) are particularly susceptible to suchaccelerated decomposition. Consequently, this invention encompassespharmaceutical compositions and dosage forms that contain little, ifany, lactose. As used herein, the term “lactose-free” means that theamount of lactose present, if any, is insufficient to substantiallyincrease the degradation rate of an active ingredient. Lactose-freecompositions of the invention can comprise excipients that are wellknown in the art and are listed, for example, in the U.S. Pharmacopeia(USP) SP (XXI)/NF (XVI). In general, lactose-free compositions compriseactive ingredients, a binder/filler, and a lubricant in pharmaceuticallycompatible and pharmaceutically acceptable amounts. Preferredlactose-free dosage forms comprise active ingredients, microcrystallinecellulose, pre-gelatinized starch, and magnesium stearate.

This invention further encompasses anhydrous pharmaceutical compositionsand dosage forms comprising active ingredients, since water canfacilitate the degradation of some compounds. For example, the additionof water (e.g., 5%) is widely accepted in the pharmaceutical arts as ameans of simulating long-term storage in order to determinecharacteristics such as shelf-life or the stability of formulations overtime. See, e.g., Jens T. Carstensen (1995) Drug Stability: Principles &Practice, 2d. Ed., Marcel Dekker, NY, N.Y., 379-80. In effect, water andheat accelerate the decomposition of some compounds. Thus, the effect ofwater on a formulation can be of great significance since moistureand/or humidity are commonly encountered during manufacture, handling,packaging, storage, shipment, and use of formulations.

Anhydrous pharmaceutical compositions and dosage forms of the inventioncan be prepared using anhydrous or low moisture containing ingredientsand low moisture or low humidity conditions. Pharmaceutical compositionsand dosage forms that comprise lactose and at least one activeingredient including a primary or secondary amine are preferablyanhydrous if substantial contact with moisture and/or humidity duringmanufacturing, packaging, and/or storage is expected.

An anhydrous pharmaceutical composition should be prepared and storedsuch that its anhydrous nature is maintained. Accordingly, anhydrouscompositions are preferably packaged using materials known to preventexposure to water such that they can be included in suitable formularykits. Examples of suitable packaging include, but are not limited to,hermetically sealed foils, plastics, unit dose containers (e.g., vials),blister packs, and strip packs.

The invention further encompasses pharmaceutical compositions and dosageforms that comprise one or more compounds that reduce the rate by whichan active ingredient will decompose. Such compounds, which are referredto herein as “stabilizer” include, but are not limited to, antioxidantssuch as ascorbic acid, pH buffers, or salt buffers.

Like the amounts and types of excipients, the amounts and specific typesof active ingredients in a dosage form may differ depending on factorssuch as, but not limited to, the route by which it is to be administeredto patients. However, typical dosage forms of the invention include acompound of the invention, or a pharmaceutically acceptable salt,solvate, clathrate, or prodrug thereof in an amount of from about 1 mgto about 1000 mg, preferably in an amount of from about 50 mg to about500 mg, and most preferably in an amount of from about 75 mg to about350 mg. The typical total daily dosage of a compound of the invention,or a pharmaceutically acceptable salt, solvate, clathrate, or prodrugthereof can range from about 1 mg to about 5000 mg per day, preferablyin an amount from about 50 mg to about 1500 mg per day, more preferablyfrom about 75 mg to about 1000 mg per day. It is within the skill of theart to determine the appropriate dose and dosage form for a givenpatient.

Oral Dosage Forms

Pharmaceutical compositions of the invention that are suitable for oraladministration can be presented as discrete dosage forms, such as, butare not limited to, tablets (e.g., chewable tablets), caplets, capsules,and liquids (e.g., flavored syrups). Such dosage forms containpredetermined amounts of active ingredients, and may be prepared bymethods of pharmacy well known to those skilled in the art. Seegenerally, Remington's Pharmaceutical Sciences (1990) 18th ed., MackPublishing, Easton Pa.

Typical oral dosage forms of the invention are prepared by combining theactive ingredient(s) in an admixture with at least one excipientaccording to conventional pharmaceutical compounding techniques.Excipients can take a wide variety of forms depending on the form ofpreparation desired for administration. For example, excipients suitablefor use in oral liquid or aerosol dosage forms include, but are notlimited to, water, glycols, oils, alcohols, flavoring agents,preservatives, and coloring agents. Examples of excipients suitable foruse in solid oral dosage forms (e.g., powders, tablets, capsules, andcaplets) include, but are not limited to, starches, sugars,micro-crystalline cellulose, diluents, granulating agents, lubricants,binders, and disintegrating agents.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit forms, in which case solidexcipients are employed. If desired, tablets can be coated by standardaqueous or nonaqueous techniques. Such dosage forms can be prepared byany of the methods of pharmacy. In general, pharmaceutical compositionsand dosage forms are prepared by uniformly and intimately admixing theactive ingredients with liquid carriers, finely divided solid carriers,or both, and then shaping the product into the desired presentation ifnecessary.

For example, a tablet can be prepared by compression or molding.Compressed tablets can be prepared by compressing in a suitable machinethe active ingredients in a free-flowing form such as powder orgranules, optionally mixed with an excipient. Molded tablets can be madeby molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

Examples of excipients that can be used in oral dosage forms of theinvention include, but are not limited to, binders, fillers,disintegrants, and lubricants. Binders suitable for use inpharmaceutical compositions and dosage forms include, but are notlimited to, corn starch, potato starch, or other starches, gelatin,natural and synthetic gums such as acacia, sodium alginate, alginicacid, other alginates, powdered tragacanth, guar gum, cellulose and itsderivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethylcellulose calcium, sodium carboxymethyl cellulose), polyvinylpyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropylmethyl cellulose, (e.g., Nos. 2208, 2906, 2910), microcrystallinecellulose, and mixtures thereof.

Suitable forms of microcrystalline cellulose include, but are notlimited to, the materials sold as AVICEL-PH-101, AVICEL-PH-103 AVICELRC-581, AVICEL-PH-105 (available from FMC Corporation, American ViscoseDivision, Avicel Sales, Marcus Hook, Pa.), and mixtures thereof. Onespecific binder is a mixture of microcrystalline cellulose and sodiumcarboxymethyl cellulose sold as AVICEL RC-581. Suitable anhydrous or lowmoisture excipients or additives include AVICEL-PH-103J and Starch 1500LM.

Examples of fillers suitable for use in the pharmaceutical compositionsand dosage forms disclosed herein include, but are not limited to, talc,calcium carbonate (e.g., granules or powder), microcrystallinecellulose, powdered cellulose, dextrates, kaolin, mannitol, silicicacid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.The binder or filler in pharmaceutical compositions of the invention istypically present in from about 50 to about 99 weight percent of thepharmaceutical composition or dosage form.

Disintegrants are used in the compositions of the invention to providetablets that disintegrate when exposed to an aqueous environment.Tablets that contain too much disintegrant may disintegrate in storage,while those that contain too little may not disintegrate at a desiredrate or under the desired conditions. Thus, a sufficient amount ofdisintegrant that is neither too much nor too little to detrimentallyalter the release of the active ingredients should be used to form solidoral dosage forms of the invention. The amount of disintegrant usedvaries based upon the type of formulation, and is readily discernible tothose of ordinary skill in the art. Typical pharmaceutical compositionscomprise from about 0.5 to about 15 weight percent of disintegrant,preferably from about 1 to about 5 weight percent of disintegrant.

Disintegrants that can be used in pharmaceutical compositions and dosageforms of the invention include, but are not limited to, agar-agar,alginic acid, calcium carbonate, microcrystalline cellulose,croscarmellose sodium, crospovidone, polacrilin potassium, sodium starchglycolate, potato or tapioca starch, other starches, pre-gelatinizedstarch, other starches, clays, other algins, other celluloses, gums, andmixtures thereof.

Lubricants that can be used in pharmaceutical compositions and dosageforms of the invention include, but are not limited to, calciumstearate, magnesium stearate, mineral oil, light mineral oil, glycerin,sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid,sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanutoil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, andsoybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, andmixtures thereof. Additional lubricants include, for example, a syloidsilica gel (AEROSIL 200, manufactured by W.R. Grace Co. of Baltimore,Md.), a coagulated aerosol of synthetic silica (marketed by Degussa Co.of Plano, Tex.), CAB-O-SIL (a pyrogenic silicon dioxide product sold byCabot Co. of Boston, Mass.), and mixtures thereof. If used at all,lubricants are typically used in an amount of less than about 1 weightpercent of the pharmaceutical compositions or dosage forms into whichthey are incorporated.

Controlled Release Dosage Forms

Active ingredients of the invention can be administered by controlledrelease means or by delivery devices that are well known to those ofordinary skill in the art. Examples include, but are not limited to,those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809;3,598,123; and 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548,5,073,543, 5,639,476, 5,354,556, and 5,733,566, each of which isincorporated herein by reference. Such dosage forms can be used toprovide slow or controlled-release of one or more active ingredientsusing, for example, hydroxypropylmethyl cellulose, other polymermatrices, gels, permeable membranes, osmotic systems, multilayercoatings, microparticles, liposomes, microspheres, or a combinationthereof to provide the desired release profile in varying proportions.Suitable controlled-release formulations known to those of ordinaryskill in the art, including those described herein, can be readilyselected for use with the active ingredients of the invention. Theinvention thus encompasses single unit dosage forms suitable for oraladministration such as, but not limited to, tablets, capsules, gelcaps,and caplets that are adapted for controlled-release.

All controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledcounterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include extended activity of the drug, reduced dosagefrequency, and increased patient compliance. In addition,controlled-release formulations can be used to affect the time of onsetof action or other characteristics, such as blood levels of the drug,and can thus affect the occurrence of side (e.g., adverse) effects.

Most controlled-release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release of otheramounts of drug to maintain this level of therapeutic or prophylacticeffect over an extended period of time. In order to maintain thisconstant level of drug in the body, the drug must be released from thedosage form at a rate that will replace the amount of drug beingmetabolized and excreted from the body. Controlled-release of an activeingredient can be stimulated by various conditions including, but notlimited to, pH, temperature, enzymes, water, or other physiologicalconditions or compounds.

A particular extended release formulation of this invention comprises atherapeutically or prophylactically effective amount of a compound ofthe invention, or a pharmaceutically acceptable salt, solvate, hydrate,clathrate, or prodrug thereof, in spheroids which further comprisemicrocrystalline cellulose and, optionally,hydroxypropylmethyl-cellulose coated with a mixture of ethyl celluloseand hydroxypropylmethylcellulose. Such extended release formulations canbe prepared according to U.S. Pat. No. 6,274,171, the entire teachingsof which are incorporated herein by reference.

A specific controlled-release formulation of this invention comprisesfrom about 6% to about 40% a compound of the invention by weight, about50% to about 94% microcrystalline cellulose, NF, by weight, andoptionally from about 0.25% to about 1% by weight ofhydroxypropyl-methylcellulose, USP, wherein the spheroids are coatedwith a film coating composition comprised of ethyl cellulose andhydroxypropylmethylcellulose.

Parenteral Dosage Forms

Parenteral dosage forms can be administered to patients by variousroutes including, but not limited to, subcutaneous, intravenous(including bolus injection), intramuscular, and intraarterial. Becausetheir administration typically bypasses patients' natural defensesagainst contaminants, parenteral dosage forms are preferably sterile orcapable of being sterilized prior to administration to a patient.Examples of parenteral dosage forms include, but are not limited to,solutions ready for injection, dry products ready to be dissolved orsuspended in a pharmaceutically acceptable vehicle for injection,suspensions ready for injection, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage forms ofthe invention are well known to those skilled in the art. Examplesinclude, but are not limited to: Water for Injection USP; aqueousvehicles such as, but not limited to, Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, and Lactated Ringer's Injection; water-miscible vehicles suchas, but not limited to, ethyl alcohol, polyethylene glycol, andpolypropylene glycol; and non-aqueous vehicles such as, but not limitedto, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate,isopropyl myristate, and benzyl benzoate.

Compounds that increase the solubility of one or more of the activeingredients disclosed herein can also be incorporated into theparenteral dosage forms of the invention.

Transdermal, Topical, and Mucosal Dosage Forms

Transdermal, topical, and mucosal dosage forms of the invention include,but are not limited to, ophthalmic solutions, sprays, aerosols, creams,lotions, ointments, gels, solutions, emulsions, suspensions, or otherforms known to one of skill in the art. See, e.g., Remington'sPharmaceutical Sciences (1980 & 1990) 16th and 18th eds., MackPublishing, Easton Pa. and Introduction to Pharmaceutical Dosage Forms(1985) 4th ed., Lea & Febiger, Philadelphia. Dosage forms suitable fortreating mucosal tissues within the oral cavity can be formulated asmouthwashes or as oral gels. Further, transdermal dosage forms include“reservoir type” or “matrix type” patches, which can be applied to theskin and worn for a specific period of time to permit the penetration ofa desired amount of active ingredients.

Suitable excipients (e.g., carriers and diluents) and other materialsthat can be used to provide transdermal, topical, and mucosal dosageforms encompassed by this invention are well known to those skilled inthe pharmaceutical arts, and depend on the particular tissue to which agiven pharmaceutical composition or dosage form will be applied. Withthat fact in mind, typical excipients include, but are not limited to,water, acetone, ethanol, ethylene glycol, propylene glycol,butane-1,3-diol, isopropyl myristate, isopropyl palmitate, mineral oil,and mixtures thereof to form lotions, tinctures, creams, emulsions, gelsor ointments, which are non-toxic and pharmaceutically acceptable.Moisturizers or humectants can also be added to pharmaceuticalcompositions and dosage forms if desired. Examples of such additionalingredients are well known in the art. See, e.g., Remington'sPharmaceutical Sciences (1980 & 1990) 16th and 18th eds., MackPublishing, Easton Pa.

Depending on the specific tissue to be treated, additional componentsmay be used prior to, in conjunction with, or subsequent to treatmentwith active ingredients of the invention. For example, penetrationenhancers can be used to assist in delivering the active ingredients tothe tissue. Suitable penetration enhancers include, but are not limitedto: acetone; various alcohols such as ethanol, oleyl, andtetrahydrofuryl; alkyl sulfoxides such as dimethyl sulfoxide; dimethylacetamide; dimethyl formamide; polyethylene glycol; pyrrolidones such aspolyvinylpyrrolidone; Kollidon grades (Povidone, Polyvidone); urea; andvarious water-soluble or insoluble sugar esters such as Tween 80(polysorbate 80) and Span 60 (sorbitan monostearate).

The pH of a pharmaceutical composition or dosage form, or of the tissueto which the pharmaceutical composition or dosage form is applied, mayalso be adjusted to improve delivery of one or more active ingredients.Similarly, the polarity of a solvent carrier, its ionic strength, ortonicity can be adjusted to improve delivery. Compounds such asstearates can also be added to pharmaceutical compositions or dosageforms to advantageously alter the hydrophilicity or lipophilicity of oneor more active ingredients so as to improve delivery. In this regard,stearates can serve as a lipid vehicle for the formulation, as anemulsifying agent or surfactant, and as a delivery-enhancing orpenetration-enhancing agent. Different salts, hydrates or solvates ofthe active ingredients can be used to further adjust the properties ofthe resulting composition.

Combination Therapy

The methods for immunosuppression or for treating or preventinginflammatory conditions and immune disorders in a patient in needthereof can further comprise administering to the patient beingadministered a compound of this invention, an effective amount of one ormore other active agents. Such active agents may include those usedconventionally for immunosuppression or for inflammatory conditions orimmune disorders. These other active agents may also be those thatprovide other benefits when administered in combination with thecompounds of this invention. For example, other therapeutic agents mayinclude, without limitation, steroids, non-steroidal anti-inflammatoryagents, antihistamines, analgesics, immunosuppressive agents andsuitable mixtures thereof. In such combination therapy treatment, boththe compounds of this invention and the other drug agent(s) areadministered to a subject (e.g., humans, male or female) by conventionalmethods. The agents may be administered in a single dosage form or inseparate dosage forms. Effective amounts of the other therapeutic agentsand dosage forms are well known to those skilled in the art. It is wellwithin the skilled artisan's purview to determine the other therapeuticagent's optimal effective-amount range.

In one embodiment of the invention where another therapeutic agent isadministered to a subject, the effective amount of the compound of thisinvention is less than its effective amount when the other therapeuticagent is not administered. In another embodiment, the effective amountof the conventional agent is less than its effective amount when thecompound of this invention is not administered. In this way, undesiredside effects associated with high doses of either agent may beminimized. Other potential advantages (including without limitationimproved dosing regimens and/or reduced drug cost) will be apparent tothose of skill in the art.

In one embodiment relating to autoimmune and inflammatory conditions,the other therapeutic agent may be a steroid or a non-steroidalanti-inflammatory agent. Particularly useful non-steroidalanti-inflammatory agents, include, but are not limited to, aspirin,ibuprofen, diclofenac, naproxen, benoxaprofen, flurbiprofen, fenoprofen,flubufen, ketoprofen, indoprofen, piroprofen, carprofen, oxaprozin,pramoprofen, muroprofen, trioxaprofen, suprofen, aminoprofen,tiaprofenic acid, fluprofen, bucloxic acid, indomethacin, sulindac,tolmetin, zomepirac, tiopinac, zidometacin, acemetacin, fentiazac,clidanac, oxpinac, mefenamic acid, meclofenamic acid, flufenamic acid,niflumic acid, tolfenamic acid, diflurisal, flufenisal, piroxicam,sudoxicam, isoxicam; salicylic acid derivatives, including aspirin,sodium salicylate, choline magnesium trisalicylate, salsalate,diflunisal, salicylsalicylic acid, sulfasalazine, and olsalazin;para-aminophenol derivatives including acetaminophen and phenacetin;indole and indene acetic acids, including indomethacin, sulindac, andetodolac; heteroaryl acetic acids, including tolmetin, diclofenac, andketorolac; anthranilic acids (fenamates), including mefenamic acid, andmeclofenamic acid; enolic acids, including oxicams (piroxicam,tenoxicam), and pyrazolidinediones (phenylbutazone, oxyphenthartazone);and alkanones, including nabumetone and pharmaceutically acceptablesalts thereof and mixtures thereof. For a more detailed description ofthe NSAIDs, see Paul A. Insel, Analgesic-Antipyretic andAntiinflammatory Agents and Drugs Employed in the Treatment of Gout, inGoodman & Gilman's The Pharmacological Basis of Therapeutics 617-57(Perry B. Molinhoff and Raymond W. Ruddon eds., 9th ed 1996) and Glen R.Hanson, Analgesic, Antipyretic and Anti-Inflammatory Drugs in Remington:The Science and Practice of Pharmacy Vol II 1196-1221 (A. R. Gennaro ed.19th ed. 1995) which are hereby incorporated by reference in theirentireties.

Of particular relevance to allergic disorders, the other therapeuticagent may be an antihistamine. Useful antihistamines include, but arenot limited to, loratadine, cetirizine, fexofenadine, desloratadine,diphenhydramine, chlorpheniramine, chlorcyclizine, pyrilamine,promethazine, terfenadine, doxepin, carbinoxamine, clemastine,tripelennamine, brompheniramine, hydroxyzine, cyclizine, meclizine,cyproheptadine, phenindamine, acrivastine, azelastine, levocabastine,and mixtures thereof. For a more detailed description of antihistamines,see Goodman & Gilman's The Pharmacological Basis of Therapeutics (2001)651-57, 10^(th) ed).

Immunosuppressive agents include glucocorticoids, corticosteroids (suchas Prednisone or Solumedrol), T cell blockers (such as cyclosporin A andFK506), purine analogs (such as azathioprine (Imuran)), pyrimidineanalogs (such as cytosine arabinoside), alkylating agents (such asnitrogen mustard, phenylalanine mustard, busulfan, andcyclophosphamide), folic acid antagonists (such as aminopterin andmethotrexate), antibiotics (such as rapamycin, actinomycin D, mitomycinC, puramycin, and chloramphenicol), human IgG, antilymphocyte globulin(ALG), and antibodies (such as anti-CD3 (OKT3), anti-CD4 (OKT4),anti-CD5, anti-CD7, anti-IL-2 receptor, anti-alpha/beta TCR,anti-ICAM-1, anti-CD20 (Rituxan), anti-IL-12 and antibodies toimmunotoxins).

The foregoing and other useful combination therapies will be understoodand appreciated by those of skill in the art. Potential advantages ofsuch combination therapies include a different efficacy profile, theability to use less of each of the individual active ingredients tominimize toxic side effects, synergistic improvements in efficacy,improved ease of administration or use and/or reduced overall expense ofcompound preparation or formulation.

Other Embodiments

The compounds of this invention may be used as research tools (forexample, as a positive control for evaluating other potential CRACinhibitors, or IL-2, IL-4, IL-5, IL-13, GM-CSF, TNFα, and/or IFN-γinhibitors). These and other uses and embodiments of the compounds andcompositions of this invention will be apparent to those of ordinaryskill in the art.

The invention is further defined by reference to the following examplesdescribing in detail the preparation of compounds of the invention. Itwill be apparent to those skilled in the art that many modifications,both to materials and methods, may be practiced without departing fromthe purpose and interest of this invention. The following examples areset forth to assist in understanding the invention and should not beconstrued as specifically limiting the invention described and claimedherein. Such variations of the invention, including the substitution ofall equivalents now known or later developed, which would be within thepurview of those skilled in the art, and changes in formulation or minorchanges in experimental design, are to be considered to fall within thescope of the invention incorporated herein.

EXAMPLES Experimental Rationale

Without wishing to be bound by theory, it is believed that the compoundsof this invention inhibit CRAC ion channels, thereby inhibitingproduction of IL-2 and other key cytokines involved with inflammatoryand immune responses. The examples that follow demonstrate theseproperties.

Materials and General Methods

Reagents and solvents used below can be obtained from commercial sourcessuch as Aldrich Chemical Co. (Milwaukee, Wis., USA). ¹H-NMR and ¹³C-NMRspectra were recorded on a Varian 300 MHz NMR spectrometer. Significantpeaks are tabulated in the order: δ (ppm): chemical shift, multiplicity(s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br s,broad singlet), coupling constant(s) in Hertz (Hz) and number ofprotons.

Manual patch clamp experiments are conducted in the tight-sealwhole-cell configuration at room temperature (21-25° C.). Patch pipettesare fashioned from borosilicate glass capillary tubes and haveresistances between 2-4 MΩ after filling with standard intracellularsolution. High resolution current recordings are acquired with acomputer-based patch clamp amplifier system (EPC-10, HEKA, Lambrecht,Germany). All voltages are corrected for a liquid junction potential of10 mV between external and internal solutions with glutamate as theintracellular anion. Currents are filtered at 2.9 kHz and digitized at10 is intervals. Capacitive currents and series resistance aredetermined and corrected before each voltage ramp using the automaticcapacitance compensation of the EPC-10.

Automated patch clamp experiments are conducted with the QPatch 16(Sophion Bioscience, Ballerup, Denmark) at room temperature (21-25° C.).Immediately following the establishment of giga-seal whole-cellconfiguration, the cells membrane potential is clamped at 0 mV. Voltageramps of 50 ms duration spanning the voltage range of −100 to +100 mVare then stimulated at a rate of 0.33 Hz. Currents are filtered at 2.9kHz and digitized at 200 is intervals. Capacitive currents and seriesresistance are determined and corrected before each voltage ramp usingthe automatic capacitance compensation.

Example 1 Synthesis of Representative Exemplary Compounds of thisInvention

General Procedure for Suzuki cross coupling: To a solution of[N-(3-bromo-4-methylphenyl)pyridin-2-amine] (95 mg, 0.36 mmol),dichloro-bis(triphenylphosphine)-palladium (II) (Pd(PPh₃)₂Cl₂, 60 mg,0.09 mmol), and2,6-difluoro-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)benzamide(195 mg, 0.54 mmol) in toluene (10 mL) were added Na₂CO₃ (2 N, 1.0 mL)and ethanol (1.0 mL). The stirred mixture was heated at 80° C. for 16hr. The solution was cooled to room temperature and diluted with H₂O (10mL) and EtOAc (10 mL). The organic phase was dried over Na₂SO₄,concentrated, and chromatographed to give the pure product in 61% yield.

2,6-Difluoro-N-(5-(5-methyl-2-(pyridin-2-yl)thiazol-4-yl)pyridin-2-yl)benzamide

To the solution of 1 (1 g, 6.57 mmol) in DCM (50 mL) were added2,6-difluorobenzoyl chloride (1.73 mL, 13.8 mmol) and pyridine (3.56 mL,12.5 mmol) at room temperature. After stirring for 60 min, the reactionsolution was concentrated to give the crude product 2. To the solutionof N,O-dimethylhydroxylamine hydrochloride (3.2 g, 32.8 mmol) in THF(100 mL) was added n-BuLi (2.5 M, 26.3 mL, 65.7 mmol) at −78° C. After 5min, the cold bath was removed. After stirring for 15 min, the reactionwas cooled back to −78° C. Then the solution of above crude product inTHF (25 mL) was added. The reaction was quenched by sat. NH₄Cl (100 mL)after stirring at −78° C. for 60 min. The mixture was extracted withEtOAc (3×100 mL), and the combined organic phases were dried over Na₂SO₄and concentrated. Column chromatography (Hexanes/EtOAc=1/1) afforded 3in 45% overall yield.

To the solution of 3 (1 g, 3.1 mmol) in THF (100 mL) was added ethylMgBr (3 M, 5.2 mL, 15.6 mmol) at −78° C. The solution was warmed up toroom temperature and stirred for 1 hr at room temperature. The solutionwas cooled back to −78° C. before it was quenched with sat. NH₄Cl (100mL). The solution was extracted with EtOAc (3×100 mL). The product 4 wasobtained by recrystallization from EtOAc/Hexanes as off white solid in85% yield.

The solution of 4 (1 g, 3.5 mmol) in THF (100 mL) withphenyltrimethylammonium tribromide (1.45 g, 3.85 mmol) was heated at 60°C. for 2 hr. Then the solution was quenched with water and extractedwith EtOAc (3×50 mL). The combined EtOAc solution was concentrated togive the crude product, which could be used directly for the next stepwithout further purification. The solution of resulting crude productwith pyridine-2-carbothioamide (0.58 g, 4.2 mmol) in EtOH was heated at70° C. overnight. The solution was diluted with water and extracted withEtOAc. Column chromatography (EtOAc) afforded compound 1 in 65% overallyield.

¹H NMR (400 MHz, CDCl₃) δ 8.70 (d, J=1.6 Hz, 1H), 8.61 (d, J=4.4 Hz,1H), 8.48-8.46 (m, 2H), 8.24 (d, J=8.0 Hz, 1H), 8.18-8.15 (m, 1H),7.83-7.78 (m, 1H), 7.52-7.42 (m, 1H), 7.34-7.30 (m, 1H), 7.04 (t, J=8.0Hz, 2H), 2.66 (s, 3H); ESMS calc'd (C₂₁H₁₄F₂N₄OS): 408.1; found: 409.3(M+H).

Compound 22,6-Difluoro-N-(4-(5-methyl-2-(pyridin-2-yl)thiazol-4-yl)phenyl)benzamide

To the solution of 1 (0.5 g, 3.35 mmol) in DCM (50 mL) were added2,6-difluorobenzoyl chloride (0.84 mL, 6.7 mmol) and pyridine (0.54 mL,6.7 mmol) at room temperature. After stirring for 60 min, the reactionsolution was concentrated. Column chromatography afforded 2 in 80%yield.

The solution of 2 (0.5 g, 1.73 mmol) in THF (100 mL) withphenyltrimethylammonium tribromide (0.68 g, 1.8 mmol) was heated at 60°C. for 2 hr. Then the solution was quenched with water and extractedwith EtOAc (3×50 mL). The combined EtOAc solution was concentrated togive the crude product, which could be used directly for the next stepwithout further purification. The solution of resulting crude productwith pyridine-2-carbothioamide (0.24 g, 1.74 mmol) in EtOH was heated at70° C. overnight. The solution was diluted with water and extracted withEtOAc. Column chromatography (Hexanes/EtOAc=1/1) afforded compound 2 in51% overall yield.

¹H NMR (400 MHz, CDCl₃) δ 8.60 (d, J=4.4 Hz, 1H), 8.24 (d, J=8.0 Hz,1H), 7.80-7.74 (m, 5H), 7.69 (s, 1H), 7.48-7.40 (m, 1H), 7.31-7.28 (m,1H), 7.03 (t, J=8.0 Hz, 2H), 2.65 (s, 3H); ESMS calc'd (C₂₂H₁₅F₂N₃OS):407.1; found: 408.3 (M+H).

Compound 32,6-Difluoro-N-(5-(5-methyl-2-(pyridin-3-yl)thiazol-4-yl)pyridin-2-yl)benzamide

The solution of 4 (1 g, 3.5 mmol), which was obtained following the sameapproach in the synthesis of Compound 1, in THF (100 mL) withphenyltrimethylammonium tribromide (1.45 g, 3.85 mmol) was heated at 60°C. for 2 hr. Then the solution was quenched with water and extractedwith EtOAc (3×50 mL). The combined EtOAc solution was concentrated togive the crude product, which could be used directly for the next stepwithout further purification. The solution of resulting crude productwith pyridine-3-carbothioamide (0.58 g, 4.2 mmol) in EtOH was heated at70° C. overnight. The solution was diluted with water and extracted withEtOAc. Column chromatography (EtOAc) afforded compound 3 in 61% overallyield.

¹H NMR (400 MHz, CDCl₃) δ 9.15 (d, J=1.6 Hz, 1H), 8.70-8.65 (m, 2H),8.49-8.46 (m, 2H), 8.27 (d, J=8.0 Hz, 1H), 8.17-8.14 (m, 1H), 7.50-7.38(m, 2H), 7.04 (t, J=8.0 Hz, 2H), 2.67 (s, 3H); ESMS calc'd(C₂₁H₁₄F₂N₄OS): 408.1; found: 409.3 (M+H).

Compound 42,6-Difluoro-N-(5-(5-methyl-2-(1-methyl-1H-pyrazol-4-yl)thiazol-4-yl)pyridin-2-yl)benzamide

The solution of 1 (0.34 g, 0.92 mmol), which was synthesized followingthe same procedure as for Compound 1, in EtOH with thiourea (0.11 g,1.44 mmol) was heated at 80° C. overnight. The solution was diluted withwater and 1N NaOH and extracted with EtOAc. Column chromatographyafforded 2 in 50% yield.

To the solution of CuBr₂ (94 mg, 0.42 mmol) in CH₃CN (3 mL) was addedt-BuONO (0.063 mL, 0.53 mmol) and the solution of 2 (0.125 g, 0.35 mmol)in CH₃CN (2 mL) at 0° C. The reaction was allowed to warm up to roomtemperature and was stirred for 1 hr before it was quenched with std.NaHCO₃ (10 mL). Column chromatography afforded 3 in 81% yield.

Compound 4 was obtained following the general Suzuki coupling conditionfrom 3 in 61% yield.

¹H NMR (400 MHz, CDCl₃) δ 8.63 (d, J=2.0 Hz, 1H), 8.46-8.43 (m, 2H),8.11-8.08 (m, 1H), 7.88 (s, 2H), 7.49-7.41 (m, 1H), 7.03 (t, J=8.4 Hz,2H), 3.96 (s, 3H), 2.58 (s, 3H); ESMS calc'd (C₂₀H₁₅F₂N₅OS): 411.1;found: 412.1 (M+H).

Compound 52,4-Difluoro-N-(5-(5-isopropyl-2-(1-methyl-1H-pyrazol-4-yl)thiazol-4-yl)pyridin-2-yl)benzamide

Compound 5 was obtained using a similar approach to that of Compound 4.

¹H NMR (400 MHz, CDCl₃) δ 9.04 (d, J=13.6 Hz, 1H), 8.58 (d, J=2.4 Hz,1H), 8.44 (d, J=8.8 Hz, 1H), 8.26-8.19 (m, 1H), 8.04-8.01 (m, 1H), 7.88(d, J=4.4 Hz, 2H), 7.10-7.05 (m, 1H), 7.00-6.94 (m, 1H), 3.96 (s, 3H),3.44-3.37 (m, 1H), 1.36 (d, J=6.8 Hz, 6H); ESMS calc'd (C₂₂H₁₉F₂N₅OS):439.1; found: 440.1 (M+H).

Compounds 6 and 72,6-difluoro-N-(4-(3-methyl-5-nicotinoylthiophen-2-yl)phenyl)benzamideand2,6-difluoro-N-(4-(3-methyl-5-(pyridin-3-ylmethyl)thiophen-2-yl)phenyl)benzamide

To a RT stirred solution of2,6-difluoro-N-(4-(3-methylthiophen-2-yl)phenyl)benzamide (a, 2 mmol) inDCM (100 mL) was added isonicotinoyl chloride hydrochloride (3 mmol).Addition of AlCl₃ followed, and the resulting mixture was stirred at RTfor 24 h. The mixture was poured over ice and extracted with DCM (2×50mL). The organic layers were concentrated and purified by columnchromatography to give2,6-difluoro-N-(4-(3-methyl-5-nicotinoylthiophen-2-yl)phenyl)benzamide(b, 0.33 g) as yellowish solid. ¹H-NMR (CDCl₃) δ ppm; ESMS calcd forC₂₄H₁₆F₂N₂O₂S: 420.1; found: 421.2 (M+H⁺).

To the solution of above solid (0.70 g) in THF (10 mL) and AcOH (0.2 mL)was added Zn powder (0.26 g), and the mixture was heated in sealed tubeat 160° C. for 24 h. The mixture was filtered and purified by columnchromatography to give2,6-difluoro-N-(4-(3-methyl-5-(pyridin-3-ylmethyl)thiophen-2-yl)phenyl)benzamide(0.50 g) as white solid. ¹H-NMR (CDCl₃) δ ppm; ESMS calcd forC₂₄H₁₈F₂N₂OS: 420.1; found: 421.2 (M+H⁺).

Compound 82,6-difluoro-N-(4-(3-methyl-5-(2-(pyridin-3-yl)ethyl)thiophen-2-yl)phenyl)benzamide

A stirred mixture ofN-(4-(5-bromo-3-methylthiophen-2-yl)phenyl)-2,6-difluorobenzamide (a, 1mmol), 3-ethynylpyridine (1 mmol), CuI (0.05 mmol), PdCl₂(PPh₃)₂ (0.05mmol) in THF (6 mL), and Et₃N (3 mL) was heated in sealed tube at 100°C. for 1 h. The mixture was filtered through celite and purified bycolumn chromatography to give a white solid (b, 46 mg).

The above solid (b, 23 mg) was dissolved in EtOAc (25 mL) andhydrogenated under a H2 balloon for 12 h. The mixture was filteredthrough celite and purified by column chromatography to give2,6-difluoro-N-(4-(3-methyl-5-(2-(pyridin-3-yl)ethyl)thiophen-2-yl)phenyl)benzamide(c, 11 mg) as white solid. ¹H-NMR (CDCl₃) δ 8.5 (m, 3H), 7.7 (d, 2H,J=9), 7.5 (m, 1H), 7.4 (d, 2H, J=9), 7.3 (m, 1H), 7.2 (m, 1H), 7.0 (t,2H, J=8), 6.58 (s, 1H), 3.0 (m, 4H), 2.24 (s, 3H) ppm; ESMS calcd forC₂₅H₂₀F₂N₂OS: 434.1; found: 435.1 (M+H⁺).

Compounds 9, 10 and 112,6-difluoro-N-(5-(2-(2-hydroxypropan-2-yl)-5-(pyridin-3-yl)thiophen-3-yl)pyridin-2-yl)benzamide,2,6-difluoro-N-(5-(2-(prop-1-en-2-yl)-5-(pyridin-3-yl)thiophen-3-yl)pyridin-2-yl)benzamideand2,6-difluoro-N-(5-(2-isopropyl-5-(pyridin-3-yl)thiophen-3-yl)pyridin-2-yl)benzamide

A stirred mixture ofN-(5-(5-bromo-2-(2-hydroxypropan-2-yl)thiophen-3-yl)pyridin-2-Y₁)-2,6-difluorobenzamide (a, 0.25 g), 3-pyridineboric acid (2mmol), and PdCl₂(PPh₃)₂ (0.10 mmol) in toluene (5 mL), EtOH (2 mL) andNaHCO₃ (aq. 1N, 5 mL) was heated to reflux for 3 h. The mixture wasdiluted with water (50 mL) and extracted with EtOAc (2×50 mL). Thecombined organic extracts were concentrated and purified by columnchromatography to give2,6-difluoro-N-(5-(2-(2-hydroxypropan-2-yl)-5-(pyridin-3-yl)thiophen-3-yl)pyridin-2-yl)benzamide(0.14 g) as brownish oil. ¹H-NMR (CDCl₃) δ 8.2-9.2 (m, 5H), 7.3-7.8 (m,5H), 7.05 (s, 1H), 7.0 (t, 2H, J=8), 1.56 (s, 6H) ppm; ESMS calcd forC₂₄H₁₉F₂N₃O₂S: 451.1; found: 452.1 (M+H⁺).

To a solution of the above oil (120 mg) in toluene (5 mL) was added TsOH(2 mg), and the mixture was heated to reflux for 1 h. The mixture wasneutralized with NaHCO₃ (aq, 50 mL) and extracted with DCM (2×50 mL).The organic extracts were concentrated and purified by columnchromatography to give2,6-difluoro-N-(5-(2-(prop-1-en-2-yl)-5-(pyridin-3-yl)thiophen-3-yl)pyridin-2-yl)benzamide(60 mg) as white solids. ¹H-NMR (CDCl₃) δ 8.89 (s, 1H), 8.5 (m, 2H), 8.4(d, 1H, J=8), 8.38 (s, 1H), 7.9 (d, 2H, J=8), 7.5 (m, 1H), 7.3 (m, 2H),7.0 (t, 2H, J=8), 5.3 (d, 2H, J=10), 1.98 (s, 3H) ppm; ESMS calcd forC₂₄H₁₇F₂N₃OS: 433.1; found: 434.1 (M+H⁺).

A solution of the above solid (50 mg) in EtOAc was hydrogenated under aH2 balloon for 2 h. The mixture was filtered through celite and purifiedby column chromatography to give2,6-difluoro-N-(5-(2-isopropyl-5-(pyridin-3-yl)thiophen-3-yl)pyridin-2-yl)-benzamide(20 mg) as white solid. ¹H-NMR (CDCl₃) δ 9.00 (s, 1H), 8.88 (s, 1H),8.2-8.4 (m, 3H), 7.9 (m, 2H), 7.4-7.5 (m, 2H), 7.18 (s, 1H), 7.0 (t, 2H,J=8), 3.3 (m, 1H), 1.3 (d, 6H, J=6) ppm; ESMS calcd for C₂₄H₁₉F₂N₃OS:435.1; found: 436.1 (M+H⁺).

Compound 12

Compounds 12-17 were synthesized in a similar manner to compounds 7 and8.

2,6-difluoro-N-(5-(2-(2-hydroxypropan-2-yl)-5-(pyridin-3-yl)thiophen-3-yl)pyrazin-2-yl)benzamide

ESMS calcd for C₂₃H₁₈F₂N₄O₂S: 452.1; found: 453.1 (M+H⁺).

Compound 132,6-difluoro-N-(5-(2-(prop-1-en-2-yl)-5-(pyridin-3-yl)thiophen-3-yl)pyrazin-2-yl)benzamide

¹H-NMR (CDCl₃) δ 9.88 (s, 1H), 8.88 (s, 1H), 8.4 (m, 2H), 7.9 (d, 1H,J=8), 7.3-7.5 (m, 4H), 7.0 (t, 2H, J=8), 5.5 (m, 2H), 1.98 (s, 3H) ppm;ESMS calcd for C₂₃H₁₆F₂N₄OS: 434.1; found: 435.1 (M+H⁺).

Compound 142,6-difluoro-N-(4-(2-(prop-1-en-2-yl)-5-(pyridin-3-yl)thiophen-3-yl)phenyl)benzamide

¹H-NMR (CDCl₃) δ 8.81 (s, 1H), 8.48 (s, 1H), 8.0 (m, 1H), 7.8 (m, 1H),7.6 (m, 2H), 7.5 (m, 3H), 7.3 (m, 2H), 7.0 (t, 2H, J=8), 5.2 (m, 2H),1.98 (s, 3H) ppm; ESMS calcd for C₂₅H₁₈F₂N₂OS: 432.1; found: 433.2(M+H⁺).

Compound 152,6-difluoro-N-(4-(2-isopropyl-5-(pyridin-3-yl)thiophen-3-yl)phenyl)benzamide

¹H-NMR (CDCl₃) δ 9.0 (m, 1H), 8.4 (m, 1H), 8.0 (m, 2H), 7.8 (m, 1H), 7.6(m, 2H), 7.4-7.5 (m, 5H), 7.0 (t, 2H, J=8), 3.3 (m, 1H), 1.3 (d, 6H,J=6) ppm; ESMS calcd for C₂₅H₂₀F₂N₂OS: 434.1; found: 435.3 (M+H⁺).

Compound 16N-(4-(2,5-di(pyridin-3-yl)thiophen-3-yl)phenyl)-2,6-difluorobenzamide

ESMS calcd for C₂₇H₁₇F₂N₃OS: 469.1; found: 470.1 (M+H⁺).

Compound 172,6-difluoro-N-(5-(2-methyl-5-(pyridin-3-yl)thiophen-3-yl)pyrazin-2-yl)benzamide

ESMS calcd for C₂₁H₁₄F₂N₄OS: 408.1; found: 409.2 (M+H⁺).

Example 2 Inhibition of IL-2 Production

Jurkat cells were placed in a 96 well plate (0.5 million cells per wellin 1% FBS medium), and then a test compound of this invention was addedat different concentrations. After 10 minutes, the cells were activatedwith PHA (final concentration 2.5 μg/mL) and incubated for 20 hours at37° C. under 5% CO₂. The final volume was 200 μL. Following incubation,the cells were centrifuged, and the supernatants collected and stored at−70° C. prior to assaying for IL-2 production. A commercial ELISA kit(IL-2 Eli-pair, Diaclone Research, Besancon, France) was used to detectproduction of IL-2, from which dose response curves were obtained. TheIC₅₀ value was calculated as the concentration at which 50% of maximumIL-2 production after stimulation was inhibited versus a non-stimulationcontrol.

Inhibition of other cytokines, such as IL-4, IL-5, IL-13, GM-CSF, TNFα,and IFN-γ, can be tested in a similar manner using a commerciallyavailable ELISA kit for each cytokine.

Test Compounds

IL-2 current inhibition I_(CRAC) current inhibition Jurkat/PHA/1% FBSCRACM1/STIM1-CHOK1 IC₅₀ % Inhibition at 500 nM  1 high moderate  2 highhigh  3 high high  8 high high  6 high high  7 high high A high high Bhigh moderate C high low D moderate  5 low  4 high low E moderate F highmoderate G high H high high  9 low 10 high moderate 11 high low I low Jhigh K low L low M low N high 15 high moderate 14 high low 16 high Ohigh High P high Q high R high S high 17 high 12 low 13 moderate T highA2 10 — A3 16 — A4 17 — A5 77 — A6 21 — A7 21 — A8 26 — A9 28 — A10 40 —A11 43 — A12 44 — Low activity: IC₅₀ > 100 High activity: 70 < % < 100Moderate activity: 50 < IC₅₀ < 100 Moderate activity: High activity:IC₅₀ < 50 50 < % < 70 Low activity: % < 50

Example 3 Manual Patch Clamp Studies of Inhibition of I_(CRAC) Currentin RBL Cells, Jurkat Cells, Cracm1/Stim1-Chok1, and Primary T Cells

In general, a whole cell patch clamp method is used to examine theeffects of a compound of the invention on a channel(s) that mediatesI_(CRAC). In such experiments, a baseline I_(CRAC) measurement isestablished within the first 70 voltage ramps, or 140 seconds, for apatched cell. Then the cells are perfused with the compound to be testedand the effect of the compound on I_(CRAC) is measured for at least anadditional 440 to 500 seconds. A compound that modulates I_(CRAC) (e.g.,inhibits) is a compound that is useful in the invention for modulatingCRAC ion channel activity.

1) RBL and Jurkat Cells

Cells

Rat basophilic leukemia cells (RBL-2H3) are grown in DMEM mediasupplemented with 10% fetal bovine serum in an atmosphere of 95% air/5%CO₂. Cells are seeded on glass coverslips 1-3 days before use.

Jurkat T cells are grown in RPMI media supplemented with 10% fetalbovine serum in an atmosphere of 95% air/5% CO₂. Cells are harvested bycentrifugation and transferred to a recording chamber just prior to eachexperiment.

Recording Conditions

Membrane currents of individual cells are recorded using the manualpatch clamp technique in the whole-cell configuration.

Intracellular Pipette Solution

The intracellular pipette solution contains Cs-Glutamate 100 mM; CsCl 20mM; NaCl 8 mM; MgCl₂ 3 mM; D-myo-Inositol 1,4,5-trisphosphate (InsP3)0.02 mM; CsBAPTA 10 mM; HEPES 10 mM; pH=7.2 adjusted with CsOH. Thesolution is kept on ice and shielded from light before the experimentsare preformed.

Extracellular Solution

The extracellular solution contains NaCl 140 mM; KCl 5.4 mM; CsCl 10 mM;CaCl₂ 10 mM; MgCl₂ 1 mM; HEPES 10 mM; Glucose 5.5 mM; at pH=7.4 adjustedwith NaOH.

Compound Treatment

Each compound is diluted from a 10 mM stock in series using DMSO. Thefinal DMSO concentration is always kept at 0.1%.

Experimental Procedure

I_(CRAC) currents are measured using 50 msec voltage ramps between −100mV to +100 mV. The voltage ramps are stimulated every 2 seconds for thefirst 70 sweeps, then every 5 seconds for the remainder of theexperiment. The membrane potential is held at 0 mV between the testramps. In a typical experiment, the peak inward currents will developwithin 50-100 seconds. Once the I_(CRAC) current is stabilized, thecells are perfused with a test compound in the extracellular solutionfor at least an additional 500 seconds.

Data Analysis

Off-line analysis with the Heka PatchMaster software is used to separatethe I_(CRAC) membrane current from the cells basal background currents.In a typical recording, InsP3 stimulated I_(CRAC) currents begin todevelop in 6 to 12 seconds after whole cell is established. Therefore,the first 1-4 voltage ramps represent the basal membrane currents in theabsence of I_(CRAC) and the average value is subtracted from allsubsequent traces. The current value at −80 mV for each ramp trace isthen measured and plotted against time. The resulting current versustime data is exported into a Microsoft Excel spreadsheet. The % I_(CRAC)inhibition in each cell is calculated by comparing the amount of currentjust prior to the compound perfusion to the amount of current after thecells has been perfused with the compound for 440-500 seconds. The IC₅₀value and Hill coefficient for each compound is estimated by fitting allthe individual data points to a single-site Hill equation.

2) Cho-K1 Cells Over-Expressing Stim1 and Either CracM1, CracM2 orCracM3

Cells

TREx™-CHO cells were transfected with human Stim1 (recombinant DNA inpcDNA4/TO/myc-His™ A with a myc epitope tag in the N-terminal) andeither CracM1, CracM2 or CracM3 (recombinant DNA in pcDNA 3.1 with a HAepitope tag in the N-terminal). Stably expressing cells were selected bygrowing the transfected cells in antibiotics for two to three weeks.Individual cell clones were isolated via serial dilution. Full lengthhuman Stim1, CracM1, CracM2 and CracM3 cDNA, TREx™-CHO cells,pcDNA4/TO/myc-His™ A and pcDNA 3.1 were purchased from Invitrogen(Carlsbad, Calif.). All cells clones are grown in Ham's F-12 mediasupplemented with 10% fetal bovine serum, penicillin 100 U/ml,streptomycin 100 μg/ml, Zeocin™ (200 μg/ml), Geneticin (500 μg/ml) andblasticidin (10 μg/ml) in an atmosphere of 95% air/5% CO₂. Stim1expression was induced with doxycycline (1 μg/ml) for 16-20 hrs. Cellswere removed from the tissue culture plates with a solution of 0.25%trypsin/0.02% EDTA and transferred to a recording chamber just prior toeach experiment.

Intracellular Pipette Solution

The intracellular pipette solution contains Cs-Glutamate 90 mM; NaCl 8mM; MgCl₂ 3 mM; CsCl 20 mM; CsBAPTA 20 mM; HEPES 10 mM; InsP3 0.02 mM;pH=7.2 adjusted with CsOH. The solution is kept on ice and shielded fromlight before the experiments are preformed.

Extracellular Solution

The extracellular solution contains NaCl 120 mM; KCl 5.4 mM; CsCl 10 mM;CaCl₂ 2 mM; MgCl₂ 1 mM; HEPES 10 mM; Glucose 5.5 mM; at pH=7.4 adjustedwith NaOH.

Patch-Clamp Recordings and Data Analysis

Experimental procedures and data analysis are identical to the aboveprocedures for R^(b1)-2H3 cells and Jurkat cells.

3) Primary T Cells

Preparation of Primary T Cells

Primary T cells are obtained from human whole blood samples by adding100 μL of RosetteSep® human T cell enrichment cocktail to 2 mL of wholeblood. The mixture is incubated for 20 minutes at room temperature, thendiluted with an equal volume of PBS containing 2% FBS. The mixture islayered on top of RosetteSep® DM-L density medium and then centrifugedfor 20 minutes at 1200 g at room temperature. The enriched T cells arerecovered from the plasma/density medium interface, then washed with PBScontaining 2% FBS twice, and used in patch clamp experiments followingthe procedure described for RBL cells.

Example 4 Automated Patch Clamp Studies of Inhibition of I_(CRAC)

1) Rbl-2H₃Cells.

Cells

RBL-2H3 are grown in DMEM media supplemented with 10% fetal bovineserum, penicillin 100 U/ml and streptomycin 100 μg/ml in an atmosphereof 95% air/5% CO₂. Cells are grown to confluence in 175 cm² tissueculture flask. On the experimental day, cells harvested with 0.25%trypsin/0.02% EDTA and resuspended in extracellular solution at densityof 5×10⁶ cells/ml

Intracellular Solution

The intracellular solution contains Cs-Glutamate 90 mM; NaCl 8 mM; MgCl₂3 mM; CsCl 20 mM; CsBAPTA 20 mM; HEPES 10 mM; InsP3 0.02 mM; pH=7.2adjusted with CsOH.

Extracellular Solution

The extracellular solution contains NMDGCl 120 mM; KCl 5.4 mM; CsCl 10mM; CaCl₂ 10 mM; MgCl₂ 1 mM; HEPES 10 mM; Glucose 5.5 mM; at pH=7.4adjusted with HCl.

Experimental Procedure

I_(CRAC) currents are measured using 50 msec voltage ramps between −100mV to +100 mV. The voltage ramps are stimulated every 3 seconds for atleast 570 seconds. The maximum I_(CRAC) current is allowed to developfor at least 135 seconds. Compounds diluted in extracellular solutionsare then applied twice, 30 seconds apart. After incubating the cellswith compound for 435 seconds, a reference solution is applied at theend of the experiment. The reference solution is a Ca²⁺ freeextracellular solution.

Data Analysis

Off-line analysis with the Qpatch software is used to plot the currentvalue at −80 mV for each ramp trace against time. The resulting currentversus time data is then exported into a Microsoft Excel spreadsheet.The I_(CRAC) membrane currents are separated from the cells basalbackground currents by either subtracting out the average membranecurrent values during the first 1-3 traces, or the average membranecurrent values obtained with the reference solution at the end of theexperiment. The % I_(CRAC) inhibition in each cell is calculated bycomparing the amount of current just prior to the first compoundaddition to the amount of current after the cells has been perfused withthe compound for at least 400 seconds.

2) Cho-K1 Cells Over-Expressing Stim1 and Either CracM1, CracM2 orCracM3.

Cells

The production of TREx™-CHO cells stably expressing recombinant humanStim1 and either CracM1, CracM2 or CracM3 cells is described above.Cells are grown to confluence in 175 cm² tissue culture flask. On theexperimental day, cells harvested with 0.25% trypsin/0.02% EDTA andresuspended in extracellular solution at density of 5−15×10⁶ cells/ml

Intracellular Solution

The intracellular solution contains Cs-Glutamate 90 mM; NaCl 8 mM; MgCl₂3 mM; CsCl 20 mM; CsBAPTA 20 mM; HEPES 10 mM; InsP3 0.02 mM; pH=7.2adjusted with CsOH.

Extracellular Solution

The extracellular solution contains NMDGCl 120 mM; KCl 5.4 mM; CsCl 10mM; CaCl₂ 1 mM; MgCl₂ 1 mM; HEPES 10 mM; Glucose 5.5 mM; at pH=7.4adjusted with NaOH.

Experimental Procedure and Data Analysis

Experimental procedures and data analysis are identical to the aboveprocedures for R^(b1)-2H3 cells.

Example 5 Inhibition of Multiple Cytokines in Primary Human PBMCs

Human peripheral blood mononuclear cells (PBMCs) were prepared fromheparinized human blood by separation over a Ficoll density gradient.

PBMCs are stimulated with phytohemagglutinin (PHA) in the presence ofvarying concentrations of compounds of the invention or cyclosporine A(CsA), a known inhibitor of cytokine production. Cytokine production ismeasured using commercially available human ELISA assay kits (from CellScience, Inc.) following the manufacturers instructions.

Alternatively, PBMCs with 10% FCS at 1-2×10⁶/ml are stimulated withpre-coated with anti-CD3 (clone UCHT1) and anti-CD28 (cloneANC28.1/5D10) at 5 μg/ml each, with or without compound or DMSO (maximumconcentration: 0.1%). Cell cultures are incubated at 37° C., 5% CO₂.Samples of the culture supernatant are collected after 48-72 hrs.incubation for measurement of multiple cytokines. Cytokines present inthe supernatants are quantified using BioRad BioPlex assays according tothe manufacturer's instructions.

The compounds of the invention are expected to be potent inhibitors ofIL-2, IL-4, IL-5, IL-13, GM-CSF, IFN-γ and TNF-α in primary human PBMcells. In addition, compounds of the invention are not expected toinhibit the anti-inflammatory cytokine, IL-10.

Example 6 Inhibition of Degranulation in RBL Cells

Procedure:

The day before the assay is performed, RBL cells, that have been grownto confluence in a 96 well plate, are incubated at 37° C. for at least 2hours. The medium is replaced in each well with 100 μL of fresh mediumcontaining 24 g/mL of anti-DNP IgE.

On the following day, the cells are washed once with PRS (2.6 mM glucoseand 0.1% BSA) and 160 μL of PRS is added to each well. A test compoundis added to a well in a 20 μL solution at 10× of the desiredconcentration and incubated for 20 to 40 minutes at 37° C. 20 μL of 10×mouse anti-IgE (10 4/mL) is added. Maximum degranulation occurs between15 to 40 minutes after addition of anti-IgE.

Compounds of the invention are expected to inhibit degranulation.

Example 7 Inhibition of Chemotaxis in T Cells

T-cell isolation:

Twenty ml aliquots of heparinized whole blood (2 pig, 1 human) aresubjected to density gradient centrifugation on Ficoll Hypaque. Thebuffy coat layers representing peripheral blood mononuclear cells(PBMCs) containing lymphocytes and monocytes are washed once,resuspended in 12 ml of incomplete RPMI 1640 and then placed ingelatin-coated T75 culture flasks for 1 hr at 37° C. The non-adherentcells, representing peripheral blood lymphocytes (PBLs) depleted ofmonocytes, are resuspended in complete RPMI media and placed in looselypacked activated nylon wool columns that have been equilibrated withwarm media. After 1 hr at 37° C., the non-adherent T cell populationsare eluted by washing of the columns with additional media. The T cellpreparations are centrifuged, resuspended in 5 ml of incomplete RPMI,and counted using a hemocytometer.

Cell Migration Assay:

Aliquots of each T cell preparation are labeled with Calcien AM(TefLabs) and suspended at a concentration of 2.4×10⁶/ml inHEPES-buffered Hank's Balanced Salt Solution containing 1.83 mM CaCl₂and 0.8 mM MgCl₂, pH 7.4 (HHBSS). An equal volume of HHBSS containing 0,20 nM, 200 nM or 2000 nM of compound 1 or 20 nM EDTA is then added andthe cells incubated for 30 min at 37° C. Fifty μl aliquots of the cellsuspensions (60,000 cells) are placed on the membrane (pore size 5 μm)of a Neuroprobe ChemoTx 96 well chemotaxis unit that have been affixedover wells containing 10 ng/ml MIP-1α in HHBSS. The T cells are allowedto migrate for 2 hr at 37° C., after which the apical surface of themembrane is wiped clean of cells. The chemotaxis units are then placedin a CytoFluor 4000 (PerSeptive BioSystems) and the fluorescence of eachwell measured (excitation and emission wavelengths of 450 and 530 nm,respectively). The number of migrating cells in each well is determinedfrom a standard curve generated from measuring the fluorescence ofserial two-fold dilutions of the labeled cells placed in the lower wellsof the chemotaxis unit prior to affixing the membrane.

Compounds of the invention are expected to inhibit chemotactic responseof T cells.

All publications, patent applications, patents, and other documentscited herein are incorporated by reference in their entirety. In case ofconflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting in any way.

1. A compound of formula (I):

or a pharmaceutically acceptable salt thereof; wherein: each of X₁ andX₂ is independently N, C or N⁺O⁻; provided that one of X₁ or X₂ is C,and the other is N or N⁺O⁻; Y₁ is S or CH; Y₂ is S, N, or CH; Z is abond or divalent radicals of alkyl, alkenyl, alkynyl, alkoxy,alkoxyalkyl, alkylaminoalkyl, cycloalkyl, heteroaryl, alkylcycloalkyl,or alkyl-substituted alkylcycloalkyl, wherein one or more carbon atomsis optionally replaced with O, S, or N; Y₃ is CH₂ or C═O; R¹ isheteroaryl, aryl, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, or (C₂-C₆)alkynyl,wherein each substituent represented by R¹ is independently andoptionally substituted with one to three halo, (C₁-C₄)alkyl,(C₂-C₄)alkenyl, (C₂-C₄)alkynyl, COR^(E), COOR⁶, CON(R⁶)₂, N(R⁶)₂,NR⁶CON(R⁶)₂, NR⁶CSN(R⁶)₂, OR⁶, S(O)_(p)R⁶, S(O)_(p)N(R⁶)₂, CN, NO₂, orN₃; R² is (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, heteroaryl,heteroaryl(C₁-C₂)alkyl, heteroaryl(C₂)alkenyl, heteroaryl(C₂)alkynyl,aryl(C₁-C₂)alkyl, aryl(C₂)alkenyl, aryl(C₂)alkynyl, CN, COR⁶, COOR⁶,CON(R⁶)₂, CSR⁶, CSOR⁶, or CSN(R⁶)₂, wherein each substituent representedby R² is independently and optionally substituted with one to threehalo, (C₁-C₄)alkyl, (C₂-C₄)alkenyl, (C₂-C₄)alkynyl, COR⁶, COOR⁶,CON(R⁶)₂, N(R⁶)₂, NR⁶CON(R⁶)₂, NR⁶CSN(R⁶)₂, OR⁶, SR⁶, CN, NO₂, or N₃; R³is H, halo, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, aryl,heteroaryl, (C₃-C₇)cycloalkyl, heterocycloalkyl, COR⁶, COOR⁶, CON(R⁶)₂,N(R⁶)₂, NR⁶CON(R⁶)₂, NR⁶CSN(R⁶)₂, OR⁶, S(O)_(p)R⁶, CN, NO₂,S(O)_(p)N(R⁶)₂ or N₃, wherein each substituent represented by R³ havinga hydrogen atom is optionally and independently substituted with halo,(C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₁-C₆)alkoxy, (C₁-C₆)haloalkoxy,(C₁-C₆)hydroxyalkyl, —OH, —NH₂, NH(C₁-C₃)alkyl, N((C₁-C₃)alkyl)₂, or CN;R⁴ is H, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, heteroaryl,heteroaryl(C₁-C₂)alkyl, heteroaryl(C₂)alkenyl, heteroaryl(C₂)alkynyl,aryl, aryl(C₁-C₂)alkyl, aryl(C₂)alkenyl, aryl(C₂)alkynyl,(C₃-C₇)cycloalkyl, heterocycloalkyl, OR⁶, or CON(R⁶)₂; each R⁵ isindependently halo, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,heteroaryl, heteroaryl(C₁-C₂)alkyl, heteroaryl(C₂)alkenyl,heteroaryl(C₂)alkynyl, (C₃-C₇)cycloalkyl, heterocycloalkyl, aryl,aryl(C₁-C₂)alkyl, aryl(C₂)alkenyl, aryl(C₂)alkynyl, (C₁-C₆)haloalkyl,COR⁶, COOR⁶, CON(R⁶)₂, N(R⁶)₂, NR⁶CON(R⁶)₂, NR⁶CSN(R⁶)₂, OR⁶,S(O)_(p)R⁶, CN, NO₂, or N₃, wherein each substituent represented by R⁵having a hydrogen atom is optionally an independently substituted withhalo, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₁-C₆)alkoxy, (C₁-C₆)haloalkoxy,(C₁-C₆)hydroxyalkyl, aryl, heteroaryl, (C₃-C₇)cycloalkyl,heterocycloalkyl, —OH, —NH₂, NH(C₁-C₃)alkyl, N((C₁-C₃)alkyl)₂, or CN;each R⁶ is independently H, (C₁-C₆)alkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, (C₃-C₇)cycloalkyl, heterocycloalkyl, heteroaryl,heteroaryl(C₁-C₂)alkyl, heteroaryl(C₂)alkenyl, heteroaryl(C₂)alkynyl,aryl, aryl(C₁-C₂)alkyl, aryl(C₂)alkenyl, or aryl(C₂)alkynyl or two R⁶substituents attached to the same or adjacent atoms are taken togetherto form a heterocycloalkyl or heteroaryl; n is 0, 1, 2, 3, 4, or 5; andp is 0, 1, or 2; wherein one but not both of Y₁ and Y₂ is S; and one ofthe dashed bonds is a double bond, and one is a single bond; when Z is abond, Y₂ is CH, X₁ is N, X₂ is C, and R² is methyl, then R¹ is otherthan 1,3-oxazol-5-yl; and when Z is a bond, Y₂ is CH, X₁ is C, X₂ is Nand R² is methyl, then R¹ is other than 1-methyl-1H-imidazol-5-yl,1,3-thiazol-2-yl and 1H-imidazol 5 yl.
 2. The compound of claim 1,wherein Z is divalent radicals of (C₁-C₆)alkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, heteroaryl, CO, COO, CON(R⁶), CS, CSO, or CSN(R⁶). 3.(canceled)
 4. The compound of claim 1, wherein Z is a bond.
 5. Thecompound of claim 1, wherein R¹ is heteroaryl.
 6. The compound of claim5, wherein R¹ is pyridinyl, pyrazolyl, oxazolyl, imidazolyl, ortetrazolyl, each of which is optionally N-substituted with (C₁-C₆)alkyl,which is optionally substituted with N(R⁶)₂.
 7. The compound of claim 1,wherein R² is (C₁-C₆)alkyl, (C₂-C₆)alkenyl, or heteroaryl, each of whichis optionally substituted with one to three halo, (C₁-C₄)alkyl,(C₂-C₄)alkenyl, (C₂-C₄)alkynyl, COR^(E), COOR⁶, CON(R⁶)₂, N(R⁶)₂,NR⁶CON(R⁶)₂, NR⁶CSN(R⁶)₂, OR⁶, SR⁶, CN, NO₂, or N₃.
 8. The compound ofclaim 1, wherein R³ and R⁴ are H.
 9. The compound of claim 1, wherein nis 2, and R⁵ is halo. 10-11. (canceled)
 12. The compound of claim 1,wherein X₁ is C, and X₂ is N.
 13. The compound of claim 1, wherein X₁ isN, and X₂ is C.
 14. The compound of claim 1, wherein Y₁ is S, and Y₂ isCH.
 15. The compound of claim 1, wherein Y₁ is S, and Y₂ is N.
 16. Thecompound of claim 1, wherein Y₂ is S.
 17. (canceled)
 18. A compound offormula (II):

or a pharmaceutically acceptable salt thereof; wherein: one of X₁ and X₂is N provided that the other is C; Y₁ is N or CH; Z is a bond; a —CH₂—group; a C₂ alkylene, alkenylene, or alkynylene group; or a carbonylgroup; R¹ is heteroaryl, aryl, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, or(C₂-C₆)alkynyl, and is optionally substituted with one to three halo,(C₁-C₄)alkyl, (C₂-C₄)alkenyl, (C₂-C₄)alkynyl, COR⁶, COOR⁶, CON(R⁶)₂,N(R⁶)₂, NR⁶CON(R⁶)₂, NR⁶CSN(R⁶)₂, OR⁶, S(O)_(p)R⁶, S(O)_(p)N(R⁶)₂, CN,NO₂, or N₃; R² is (C₁-C₆)alkyl, (C₂-C₆)alkenyl, or (C₂-C₆)alkynyl, andis optionally substituted with one to three halo, (C₁-C₄)alkyl,(C₂-C₄)alkenyl, (C₂-C₄)alkynyl, COR⁶, COOR⁶, CON(R⁶)₂, N(R⁶)₂,NR⁶CON(R⁶)₂, NR⁶CSN(R⁶)₂, OR⁶, SR⁶, CN, NO₂, or N₃; R³ is H, halo,(C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, COR⁶, COOR⁶, CON(R⁶)₂,N(R⁶)₂, NR⁶CON(R⁶)₂, NR⁶CSN(R⁶)₂, OR⁶, S(O)_(p)R⁶, CN, NO₂,S(O)_(p)N(R⁶)₂ or N₃, and is optionally and independently substitutedwith halo, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₁-C₆)alkoxy,(C₁-C₆)haloalkoxy, (C₁-C₆)hydroxyalkyl, —OH, —NH₂, NH(C₁-C₃)alkyl,N((C₁-C₃)alkyl)₂, or CN; R⁴ is H, (C₁-C₆)alkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, OR⁶, or CON(R⁶)₂; each R⁵ is independently halo,(C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, COR⁶, COOR⁶, CON(R⁶)₂,N(R⁶)₂, NR⁶CON(R⁶)₂, NR⁶CSN(R⁶)₂, OR⁶, S(O)_(p)R⁶, CN, NO₂, or N₃, andis optionally substituted with halo, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl,(C₁-C₆)alkoxy, (C₁-C₆)haloalkoxy, (C₁-C₆)hydroxyalkyl, —OH, —NH₂,NH(C₁-C₃)alkyl, N((C₁-C₃)alkyl)₂, or CN; each R⁶ is independently H,(C₁-C₆)alkyl, (C₂-C₆)alkenyl, or (C₂-C₆)alkynyl; n is 0, 1, 2, 3, 4 or5; and p is 0, 1 or
 2. 19. (canceled)
 20. A pharmaceutical composition,comprising a pharmaceutically acceptable carrier and a compound ofclaim
 1. 21-29. (canceled)